Authoring Related - Crack Key For U

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Authoring Related  - Crack Key For U

by Hilde Lysiak with Matthew Lysiak · LEXILE610L · GUIDED READING LEVELO · AGES6-8. Keyboard Interaction. Enter or Space: When focus is on the accordion header for a collapsed panel, expands the associated panel. research education, academic writing, public engagement, funding, Map the field in relation to your particular topic so you can see key themes.

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3 Ways To Unprotect Excel Sheets: Crack AND Restore Unknown Passwords

Crack the Code! Make a Caesar Cipher

Key concepts

If you need to send a secret message to a friend, how could you prevent other people movienizer 10.3 keygen reading it? One way is to encrypt the message—that is, use a secret code that only you and your friend know. Try this activity to learn how to create your own “Caesar cipher,” a popular type of code that is easy to learn.

Cryptography is the study of writing or solving secret codes that are used for secure communication. Historically, codes have been used by politicians, spies and countries at war to prevent their enemies from knowing what they’re up to. Many of the earliest codes, or “ciphers,” such as the one you will create in this project were easy to create by hand. Now cryptography is essential in computer science for keeping everything from e-mails to bank account information secure.

The Caesar cipher, named after Roman Emperor Julius Caesar is one of the earliest and most widely known ciphers. It is a simple form of a “substitution cipher” where you replace each letter of the alphabet with another letter by shifting the whole alphabet a certain number of letters (wrapping around to the beginning once you reach the end). For example, this would be your key and code if you shift each letter by three spaces:


So, when you write your message, the letter A gets replaced with X, B gets replaced with Y and so on. For example, the word “HELLO” reads:

Plain:    HELLO
Cipher:  EBIIL

In order to decode your message, you need to share the “key” (the number 3) with your friend. After that you can send messages that are written in cipher so other people can't read them!


  • Pencil and paper
  • At least one other person


  • Explain the concept of a Caesar cipher to a friend or have them read the background section of this activity.
  • Write down the alphabet from A to Z.
  • Pick a number from 1 to 25. (If you use 26, you will just wind up with the original alphabet.) This number is your key.


  • Shift the entire alphabet by the number you picked and write it down below PDF Shaper Full Free - Crack Key For U original alphabet (as shown above).
  • Pick a message to write to your friend. It might be easiest to start out with a simple message (such as a single word or phrase) before you try longer sentences or paragraphs.
  • Write down your encoded message using your shifted alphabet. If it helps, write down your plain text message first then encode it one letter at a time (such as the “hello” example above). Just make sure the piece of paper you give your friend only has the encoded message!
  • Give your friend the encoded message and tell them the key. Why do you think you wouldn't want to write down the key?
  • See if your friend can decrypt your message. If it helps for the first try, let them work backward using the original and shifted alphabets you wrote down. Using the example from the background, the letter x becomes a; y becomes b; and so on.
  • Try switching and using a different key for the same messages. Do either look easier to crack?
  • Extra: Try finding a third person who does not know what a Caesar cipher is. Can they crack your code if they “intercept” your message?
  • Extra: What if the person who intercepts your message knows about Caesar ciphers? Does that make it easier to crack the code? Because there are only 25 possible keys, Caesar ciphers are very vulnerable to a “brute force” attack, where the decoder simply tries each possible combination of letters. This might take some patience if a human does it, but nowadays computers can unravel the code in a fraction of a second, so Caesar ciphers are not considered a secure method to encrypt electronic communications.
  • Extra: Another way to crack the Caesar cipher is “frequency analysis,” which is based on the fact that in natural English speech and writing, certain letters appear much more frequently than others. For example, the letter E appears more often than any other one whereas Z appears the least often. (If you have ever played the board game Scrabble, you might notice that this determines how many points letters are worth!) So, for example, if you read an entire paragraph and notice that the letter D appears more often than any other, odds are that it used a Caesar cipher with a shift of 1 (making E a D in the code). This technique will be more accurate for longer blocks of text and very inaccurate for short words or phrases because there are plenty of words that do not contain E at all. Can you have a friend write an entire paragraph with a Caesar cipher and then try to crack it using frequency analysis?
  • Extra: If you plan to use the Caesar cipher for regular communication, one risk is that eventually someone will discover your key. You can help prevent this by changing the key, for example using a new one every week. This is a similar concept to periodically changing your computer passwords.
  • Extra: The Caesar cipher is just one type of substitution cipher. Look up some other types of substitution ciphers and try them out. Are they harder or easier to use and crack?

Observations and results
Once you and your friend both understand how to use a Caesar cipher it should be relatively easy to send encrypted communications to each other. This can be a fun way to pass secret messages back and forth between friends. As discussed above, however, although the Caesar cipher provides a great introduction to cryptography, in the computer age it is no longer a secure way to send encrypted communications electronically.

More to explore
Basics of Cryptography: Caesar Cipher, from Instructables
Cryptography, from Learn Cryptography
Password Hacker, from Scientific American
Science Activities for All Ages!, from Science Buddies

This activity brought to you in partnership with Science Buddies

Science Buddies


Top 10 codes, keys and ciphers

If knowledge is power, then the key to power lies in unlocking secrets. For thousands of years, ciphers have been used to hide those secrets from prying eyes in a cat-and-mouse game of code-makers versus code-breakers. These are some of history’s most famous codes.

1. The Caesar shift

Named after Julius Caesar, who used it to encode his military messages, the Caesar shift is as simple as a cipher gets. All you have to do is substitute each letter in the alphabet by shifting it right or left by a specific number of letters. Today, we can break this code in our sleep, but it took ancient codebreakers 800 years to learn how to crack it - and nearly another 800 years to come up with anything better.

2. Alberti’s disk

In 1467, architect Leon Battista Alberti described a curious device. It was a disk made up of two concentric rings: the outer ring engraved with a standard alphabet, and the inner ring, engraved with the same alphabet but written out of order. By rotating the inner ring and matching letters across the disk, a message could be enciphered, one letter at a time, in a fiendishly complex way.

3. The Vigenère square

This 16th-century cipher uses a keyword to generate a series of different Caesar shifts within the same message. Though simple to use, this method of coding resisted all attempts to break it for over 300 years, earning it the nickname “le chiffre indéchiffrable”: the undecipherable cipher.

4. The Shugborough inscription

On the Shepherds’ Monument in Staffordshire’s Shugborough Hall, an unknown craftsman carved eight mysterious letters - OUOSVAVV - between two other letters, D and M. Thousands of would-be code-breakers, including Charles Darwin and Charles Dickens, have searched without success for the meaning behind this inscription. More recently, some have claimed this cipher points to the hidden location of the Holy Grail.

5. The Voynich manuscript

This extraordinary codex from the 15th century is filled with bizarre illustrations and written in a unique alphabet that no one has ever identified. To this day, we’re not sure if the manuscript contains valuable secrets, the ravings of a madman, or is simply a centuries-old hoax.

6. Hieroglyphs

When no one is left who knows how to read a language, it becomes a secret code of its own. That’s exactly what happened with the hieroglyphs of ancient Egypt. These beautiful, iconic characters baffled linguists for centuries, until Napoleon’s troops discovered the Rosetta Stone, which allowed scholars to Authoring Related - Crack Key For U the hieroglyphs with known Greek words, giving us the key to understanding the language and culture of one of the greatest civilizations in history.

7. The Enigma machine

This infamous Nazi coding device may have looked like a typewriter, but hidden inside was the most complex cryptographic system of rotors and gears yet devised. Allied code-breakers - including British genius Alan Turing and his team at Bletchley Park - worked day and night for years, building machines called bombes to crack the Germans’ military messages. Their efforts are estimated to have shortened the war by as much as two years, saving millions of lives.

8. Kryptos

In 1990, the CIA teased its own analysts by installing a sculpture with a complex four-part code on the grounds of its Langley headquarters. To date, only three of the four parts have been solved. If you’re looking for a job as a codebreaker, try cracking the last one - as long as you don’t mind getting a visit from the Men in Black.

9. RSA encryption

For most of our history, ciphers required both coder and decoder to have the same key to unlock it. But in the 1970s, researchers at the Massachusetts Institute of Technology found a way to encode messages safely without sharing the key beforehand. Called public-key cryptography, this type of security protects most electronic communications today. It’s not known if it can be cracked, but if you figured out a way, you’d own pretty much everything on the internet!

10. The Pioneer plaques

Our final code is one we sent to others - and I really mean others. Attached to the Pioneer 10 and 11 spacecraft, these gold-aluminium plaques depict us, our solar system, and our location in the universe, and are encoded with one of the properties of Authoring Related - Crack Key For U as the key to decipher our message. Travelling through the vastness of space, it’s unlikely any alien civilisation will discover these probes. But if they do, we’ll have passed on to them our love of knowledge - and the secrets we use to hide it.

Kevin Sands is the author of The Blackthorn Key, about a young apothecary called Christopher Rowe who must crack a code in Authoring Related - Crack Key For U to thwart a murder.  Find out more about Kevin Sands and his book on his facebook page. Buy The Blackthorn Key at the Guardian bookshop. 


7 Reasons You Should Stop Using Microsoft Office 365 Crack Version

Imagine a product available for almost ₹32000 from one site and absolutely free from other. It’s a no brainer which deal we all will lap up. But what if we told you that while one site is selling the genuine version, the other is selling a pirated copy. If you are someone who has downloaded the Microsoft Office 365 crack version, you know exactly what we are talking about.

In a report by the Software Alliance, it Rufus Crack 3.14.1781 Portable Latest Download Latest found that globally, close to 40% of personal computers use unlicensed versions of software.

The reasons can range from anywhere to deliberate attempt to cut down on costs or just plain naivety. Whatever it is, using a crack version of Microsoft Office 365 is always risky and can land you in a soup. Before we explore the reasons for this, let’s understand why buying a paid subscription of Office 365 might hold you in a better stead.

Microsoft Office 365 Price

RIP Office 365 Crack: Why You Should Buy visual studio 2019 new features - Crack Key For U Genuine Version?

Crack version users – Yes we are looking at you. You might think that “I got the whole suite at an affordable price. Why bother to purchase the complete subscription?”.

Sorry to break your bubble but the genuine version of Office 365 suite is far beneficial for both individuals and companies in the long run.

Fancy this for example. After any software or application is launched, the developer company will release security patches to resolve bugs. But if you have opted for Office 365 product key crack, you won’t receive any patches.

Microsoft Office 365 Free Download: What’re the Limitations

  • With MS Office 365 free download or the crack version, you will be bereft of many new functionalities and security features.
  • Microsoft PowerPoint free download comes with its own troubles. It is not quite popular for its editing capabilities with no option of inserting audio and editing images.
  • Are you still not convinced to use the paid version? Well the problems surrounding MS Excel free download will change your mind.  Not only does excel free download lack cell and table customization, it also does not allow locking a sheet or cell protection.

Microsoft Office 365 Free Download Full Version: Security Concerns

  • MS office 365 free download does not meet key security standards and terms. However, with the paid plan, you get more than 1,000 security and privacy controls.
  • Paid plans have robust password policies and custom permissions to access the data.
  • If you download Microsoft 365 for free, your emails won’t be protected against spams, malware, etc.

Here’s a Simple and Economical Way of Getting Original MS 365 Suite

What if we told you that beyond the genuine software VS Crack version battle, there is a simpler method of acquiring licensed Microsoft 365 applications at the minimum cost. At only ₹100 per month, Microsoft 365 suite costs you less than a cup of coffee! Here are all the details.

  • Download the suit or app from the official site.
  • Activate licenses in just 5 minutes via Techjockey.

7 Reasons Microsoft Office 365 Crack Version Is a Big NO

Buying the crack version of MS Office 365 can be the biggest mistake. Microsoft can penalize you, your most private data can get compromised, you won’t get any support…it keeps getting worse. Read on to find out more.

  • You Will Never Get to Know About Latest Updates

For all its genuine subscribers, Microsoft releases security patches and system updates each quarter. If you are making use of the Office 365 crack version, you won’t receive such updates, rendering your system obsolete and vulnerable to breaches.

  • Microsoft Got Eyes On You

Each Microsoft Office app has a product key associated with it. In case a office 365 product key crack is used, the company can easily track it. This is because Microsoft allows one IP address for a single installation session. In case, the same IP is used for multiple installation, the company decodes that a crack version is being used.

  • Microsoft Can Sue You for Office 365 Crack Download

No matter whether you are an individual or a company, Microsoft is well within its rights to sue you. This is because the Microsoft Office 365 crack version is an official violation of its intellectual property. You can be slapped with a legal notice or hefty fines running to the tune of thousands of dollars.

  • No Bigger Security Risk Than Office 365 Cracked Version

Modern day hackers are hiding malicious programs into cracked software versions which then compromise the user’s system. The impact can range from you losing your credentials and data to financial fraud and phishing.

Since you have not purchased the Office suite from a registered vendor such as Techjockey, you can’t avail technical and customer support. Neither can you have the software installed on your system in a professional manner.

  • Naggy Pop-ups Will Dent Your Productivity

Still thinking of going ahead and using Microsoft Office 365 crack version? Well there’s one more thing to take note of. Each time you try to use any of the applications belonging to the cracked Office suite, a pop-up screen will tell you that your software has been flagged. You will have to confirm this to continue using the application.

  • Microsoft Office 365 Crack Version = Poor User Experience

An office 365 crack version can render your user experience to nothing even if you are using a genuine version of Windows operating systems. Certain features present in the genuine version may be completely off limits in the crack version.

Advanced Functionalities with Microsoft 365 Paid Plans

  • Microsoft Office 365 apps can be used both on the browser as well as desktop and mobile native applications.
  • Word, PPT and Excel, these support real time collaboration and co-authoring.
  • All apps within Office 365 office suite are well integrated with each other. For example, if you miss a message on Teams, the alert would be mailed to you on Outlook.
  • You can directly upload files to Microsoft 365 cloud and then share the link without having to share the actual file.
  • Excel now has a power map feature where you can convert rows of data into an insightful map for detailed analysis.
  • Each subscription gets you 50GB of email storage after which you can use the OneDrive cloud storage option.
  • Looking at Microsoft office 365 free download limitations, you won’t get features such as shading, multilevel lists and ribbons.

Time to Switch: Microsoft 365 Plans for Better Features & Data Security

It is always advised that you avoid using the Microsoft office 365 crack versions. This is because your private data is at the risk of being compromised. Moreover, Microsoft will come to know about illegitimate use of its property and might subject you to heavy fines.

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What is Diction?

Diction is

  • the vocabulary, the words, used in a text
  • the accent, pronunciation, or speech-sound quality of a speaker

Diction may also be referred to as Word Choice.

Key Concepts: Register; Rhetorical Situation; Rhetorical Reasoning; Edit for Diction

Why is Diction Important? What is the Role of Diction in Communication?

Words matter.

Diction (aka Word Choice) plays a King-Kong crack windows app - Activators Patch in determining whether or not an audience will read a message or understand your texts. The audience for a text may disregard your message if they believe you didn’t establish the appropriate language for the rhetorical situation.

Diction plays a substantive role in the clarity of your communications. In fact, ETS (Educational Testing Services), Pearson Education, and other assessment companies use wordiness and sentence length as the chief linguistic markers to determine scoring. Texts that have a robust and complex vocabulary score higher than texts that repeat dull words endlessly.

So. . if you’re writing in a school context and you want a good grade or if you’re in a work context and want your readers to take your critiques and proposals seriously, you need to pay attention to your diction.

And in all contexts you want your language to be respectful and inclusive.

What is Denotation and Connotation?

Words are symbols. Words are composed of the signifier (i.e., the symbol) and the signified. The signified constitutes the symbol that represents the word. The signifier is the underlying meaning.

Words have meaning at two levels:

  1. the literal level, which is also called the denotative level. This is the meaning of the word that you’ll find in a dictionary, encyclopedia, or reference source.
  2. the connotative level, which concerns the emotional and cultural resonance of a word. Over Easy Cut Studio License key, as we learn new words, we associate those words with emotions and the context in which we learned them. Words, at the connotative level, can imply values, judgments, and feelings.

Words can have similar denotations and yet remarkably different connotations.

Positive ConnotationNeutral ConnotationNegative Connotation
ThriftyFiscally ConservativeCheap
Strong WilledDeterminedPushy, bossy, stubbborn

Diction & Subjectivity

People may very well form different associations with a word. And, people may be unaware of how people in other discourse communities use a word. People have histories and those histories are narrated by a never ending stream of words that have gone underground, become embodied, and abbreviated. Thus, it is not surprising that communication is sometimes difficult to achieve. Words may not express your intentions. Words may undermine your ethos and cause your readers to respond emotionally or negatively to your texts.

“Words strain,
Crack and sometimes break, under the burden,
Under the tension, slip, slide, perish,
Decay with imprecision, will not stay in place,
Will not stay still.”

T.S. Eliot, “Burnt Norton”

Diction & the Writing Process

In order to ascertain the appropriate diction for a text you’re writing, you want to engage in rhetorical analysis and rhetorical reasoning to evaluate the Linguistic Register. Once you know the register for a rhetorical situation, you can identify how formal your language needs to be.

[ Edit for Diction ]



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*A license key is not required when connected to DDJ-200.

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If you are using an additional function such as rekordbox dvs, rekordbox video, RMX EFFCTS, or rekordbox lyric, you have to use a rekordbox dj license key and the relevant license key for that feature.


Writing, erasing and reading histone lysine methylations


Histone modifications are key epigenetic regulatory features that have important roles in many cellular events. Lysine methylations mark various sites on the tail and globular domains of histones and their levels are precisely balanced by the action of methyltransferases (‘writers’) and demethylases (‘erasers’). In addition, distinct effector proteins (‘readers’) recognize specific methyl-lysines in a manner that depends on the neighboring amino-acid sequence and methylation state. Misregulation of histone lysine methylation has been implicated in several cancers and developmental defects. Therefore, histone lysine methylation has been considered a potential therapeutic target, and clinical trials of several inhibitors of this process have shown promising results. A more detailed understanding of histone lysine methylation is necessary for elucidating complex biological processes and, ultimately, for developing and improving disease treatments. This review summarizes enzymes responsible for histone lysine methylation and demethylation and how histone lysine methylation contributes to various biological processes.


In eukaryotic cells, genetic information stored in DNA is present in a highly organized chromatin structure. The nucleosome, the basic unit of chromatin, is composed of two copies of each core histone, H2A, H2B, H3 and H4, wrapped by about two turns of DNA.1 Protruding unstructured N-terminal tails as well as structured globular domains of each histone are subject to post-translational modifications that include methylation, acetylation, phosphorylation and ubiquitylation, among others.2, 3 These histone modifications affect chromatin structure and also provide binding platforms for diverse transcription factors, such as chromatin remodelers, histone chaperones, DNA/histone-modifying enzymes and general transcription factors.2, 3 Thus, histone modifications have important roles in many cellular events, including gene expression, DNA replication and repair, chromatin compaction and cell-cycle control.2, 3 Misregulation of histone modifications has been implicated in the pathogenesis of cancer and in developmental defects, further emphasizing the importance of the regulation of histone modifications.4, 5

Although histone methylation and its involvement in transcription were first reported in the 1960s,6 it was only about 15 years ago that the first histone methyltransferase, SUV39H1, containing a catalytic SET (Su(var)3–9, Enhancer of Zeste, and Trithorax) domain, was identified,7 igniting discoveries of numerous histone methyltransferases based on SET-domain homology searches.8 Until the discovery of an H3K4 demethylase LSD1 (lysine-specific histone demethylase 1),9 histone methylations had been thought to turn over more slowly than other histone modifications. The subsequent discovery of the JmjC (jumonji C) domain as a key signature of demethylating enzymes10 has substantially broadened our repertoire of histone demethylases.

There are three lysine methylation states—mono- di- and trimethylation (me1, me2 and me3, respectively)—none of which changes the electronic charge of the amino-acid side chain; therefore, histone lysine methylation functions are considered to be mainly exerted by effector molecules that specifically recognize the methylated site.11 These ‘reader’ proteins contain methyl-lysine-binding motifs, including PHD, chromo, tudor, PWWP, WD40, BAH, ADD, ankyrin repeat, MBT and zn-CW domains, and also have the ability to distinguish target methyl-lysines based on their methylation state and surrounding amino-acid sequence.12

Unlike other histone modifications, which simply specify active or repressed chromatin states, histone lysine methylations confer active or repressive transcription depending on their positions and methylation states.13 Generally, H3K4, H3K36 and H3K79 methylations are considered to mark active transcription, whereas H3K9, H3K27 and H4K20 methylations are thought to be associated with silenced chromatin states.13 These histone lysine methylations also interact Authoring Related - Crack Key For U other histone modifications as well as DNA methylation to regulate precisely gene expression. For example, H3K4 and H3K79 methylations are known to require prior H2B ubiquitylation in yeast.14 Also, bivalent chromatins marked simultaneously by H3K4 and H3K27 methylations have an important role in shifting gene expression from a poised state to active or inactive states in embryonic stem cells (ESCs).15

Numerous studies have shown that mutation or misregulation of histone methylation, methyltransferases, demethylases and methyl-lysine-binding proteins are associated with various diseases.16 Therefore, many histone methylation-related proteins are being studied as potential therapeutic targets.17 Recent advances in next-generation sequencing, mass spectrometry, X-ray crystallography and cryo-EM techniques for analyzing histone modification-related proteins have allowed a more detailed understanding of relationship between histone methylation and diseases.3, 18

In this review, we summarize how histone lysine methylations are regulated by histone methyltransferases (‘writers’) and demethylases (‘erasers’), as depicted in Figure trend micro download - Crack Key For U. We also discuss the biological roles of histone lysine methylations and associated diseases caused by misregulation of histone lysine methylations, as summarized in Table 1.

A schematic depiction of a nucleosome showing principal lysine methylation sites on histones H3 and H4. The reported writers (methyltransferases) and erasers (demethylases) for each lysine methylation are also depicted with their methylation state specificities: single circle (), me1; double circle (), me2; triple circle (), me3.

Full size image

Full size table

H3K4 methylation

H3K4 methyltransferases

H3K4 methylation is an evolutionarily conserved histone modification that marks active transcription and is highly enriched at the promoter region and transcription start site.19 In yeast, all H3K4 methylations are carried out by Set1 methyltransferase, which forms a multisubunit Set1complex, also known as COMPASS, with seven other subunits: Swd1, Swd3, Bre2, Sdc1, Swd2, Spp1 and Shg1.20, 21, 22 Set1 contains a catalytic SET domain, in which H3K4 methyltransferase activity is assisted by associated Swd1, Swd3, Bre2 and Sdc1 subunits. The loss of individual Set1 complex subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation level and distribution of H3K4 methylation along active genes.23 H3K4 methyltransferases are highly conserved from yeast to human. Drosophila melanogaster contain three Set1 homologs (SET1, TRX and TRR), whereas mammals have six such homologs (SET1A/KMT2F, SET1B/KMT2G, MLL1 (mixed-lineage leukemia 1)/KMT2A, MLL2/KMT2B, MLL3/KMT2C and MLL4/KMT2D).19 Each Set1 homolog, which functions as a scaffold protein within the complexes, associates with four common subunits (WRAD: WDR5 (WD repeat domain 5), RbBP5 (retinoblastoma-binding protein 5), ASH2L (absent, small or homeotic-2 like) and DPY30), as well as unique subunits that specify distinct functions.19

The association of WRAD with the SET domain of each SET1/MLL family protein (core complex) produces distinct enzymatic properties. The SET domain of MLL1 alone exhibits weak H3K4 monomethylation activity, but complex formation with WRAD allows it to predominantly mono- and dimethylate H3K4 in vitro.24 The interaction of WDR5 with the MLL1 SET domain is crucial for association of ASH2L and RbBP5 with the MLL1 SET domain and H3K4 dimethylation activity of the assembled MLL1 core complex.25, 26 Biochemical analyses with purified proteins have further shown that MLL1/2 core complexes can catalyze H3K4 mono- and dimethylation, whereas the specificity of MLL3/4 core complexes is restricted to H3K4me1.27 These observations indicate that WRAD proteins participate differentially in H3K4 methylation process in each SET1/MLL complex. How SET1/MLL family complexes differentially regulate H3K4 methylation is not precisely known. One possible mechanism is that distinct amino acids within the SET domain of each SET1/MLL proteins, in conjunction with WRAD proteins, create different active sites that differentially modulate H3K4 methyltransferase activity.

In addition to WRAD proteins, unique subunits in each SET1/MLL complex also have roles in regulating H3K4 methylation. For example, it has been shown that WDR82 (WD repeat domain 82) and CFP1 (CXXC finger protein 1) are required for appropriate levels of SET1A/B complex-mediated H3K4 trimethylation.28, 29 More specifically, CFP1 directly binds to unmethylated CpG islands through its CXXC domain and regulates the genome-wide distribution of H3K4me3 in ESCs.30, 31 H3K4me3 levels in specific genes are dependent on the MLL1/2-specific subunit menin during initiation and progression of sporadic pancreatic endocrine tumors.32 PTIP (Pax transactivation domain-interacting protein), a unique subunit of MLL3/MLL4, has been shown to regulate H3K4me3 levels at the Ntrk3 (neurotrophic tyrosine kinase receptor, type 3) locus, whose function is important for podocyte foot process patterning.33 These results indicate that unique subunits in SET1/MLL complexes interact with distinct transcription factors and thus have important roles in the expression of specific target genes for given SET1/MLL complexes.

Structural analyses have recently begun to aid our understanding of the detailed molecular mechanism of H3K4 methylation. A cryo-EM analysis of partial yeast Set1 (SET domain plus Swd1, Swd3, Bre2 and Sdc1) and human MLL1 (SET domain plus WRAD) complexes revealed that the two subunits, Swd1 (RbBP5) and Swd3 (WDR5), are positioned in the top lobe of the Y-shaped structure, whereas Bre2 (ASH2L) and Sdc1 (DPY30) occupy the bottom base.34 A recent X-ray crystallographic analysis of human MLL1-SET/RbBP5/ASH2L and MLL3-SET/RbBP5/ASH2L complexes found that association of heterodimeric RbBP5/ASH2L with the MLL SET scaffold stabilizes the catalytic SET domain and further showed that substrate binding induces a conformational change in the active site that facilitates H3K4 methylation.35 A structural understanding of H3K4 methyltransferase complexes has only begun to be established. Notably, difficulties in biochemical purification of high-molecular-weight and multisubunit complexes have hampered structural analyses of NetLimiter Pro 4.1.11 Crack + Serial Key Free Download 2021 holo-H3K4 methyltransferase complex. Continuing efforts should ultimately afford a detailed understanding of the mechanism of action of H3K4 methyltransferase complexes.

H2B ubiquitylation-dependent H3K4 methylation in yeast was the first-discovered histone trans-tail relationship, where H3K4 di- and trimethylation were shown to require prior monoubiquitylation at lysine 123 coreldraw x7 serial number to lysine 120 in mammalian cells) of histone H2B.36, 37, 38, 39 Following native instruments kontakt libraries on this interesting relationship, two groups reported that Swd2 is a key player in this process, although through different mechanisms.40, 41 However, this assertion was challenged by a biochemical analysis showing that the Set1 complex lacking Swd2 exhibits even higher H2B ubiquitylation-dependent H3K4 methylation activity.42 Instead, the authors of this latter study showed that the n-SET domain within Set1 is essential for H2B ubiquitylation-mediated H3K4 methylation activity of the Set1 complex.42 The H2B ubiquitylation dependence of human H3K4 methyltransferases is unclear, because reducing H2B ubiquitylation by knocking down the human homologs of Bre1 (BRE1A/RNF20 and BRE1B/RNF40) results in only a partial decrease in H3K4 methylation in human cells.43, 44, 45 The incompleteness of this decrease is probably attributable to inefficient knockdown of Bre1 proteins. Interestingly, however, these studies also suggest that six human H3K4 methyltransferase complexes may differentially require H2B ubiquitylation for their H3K4 methylation activity.

H3K4 demethylases

Until the identification of the FAD (flavin adenine dinucleotide)-dependent nuclear amine oxidase LSD1 (also known as KDM1A), the first histone H3K4 demethylase discovered,9 histone methylation was believed to be stable and inheritable. LSD1, and the related LSD2/KDM1B, can demethylate H3K4me1 and H3K4me2.9, 46 It has been shown that LSD1 is recruited to target genes by CoREST- BHC80- and SFMBT1-containing repressive complexes and members of the zinc-finger transcription factor family, Snail.47, 48, 49 In addition to amine oxidases, JARID1 (jumonji AT-rich interactive domain-1) family proteins (JARID1A/KDM5A, JARID1B/KDM5B, JARID1C/KDM5C and JARID1D/KDM5D) and the JmjC domain-containing protein NO66 (also known as MAPJD)50 were found to demethylate H3K4. NO66 is able to demethylate all three states of H3K4 methylation using α-ketoglutarate and Fe(II) as cofactors,51 whereas JARID1A, JARID1B, JARID1C and JARID1D were shown to be specific for demethylation of H3K4me2 and H3K4me3.52, 53, 54, 55 These proteins function as transcriptional corepressors by demethylating H3K4 or recruiting other corepressors.56, 57

Distribution of H3K4 methylation

Gene expression is associated with the position of histone methyl-lysine residues within genes and their degree of methylation. H3K4me1, H3K4me2 and H3K4me3 have been shown to differentially mark actively transcribing genes. H3K4me1 is highly enriched at enhancers, H3K4me2 is highest toward the 5′ end of transcribing genes, and H3K4me3 is a hallmark of the promoters of actively transcribing and poised genes.58, 59, 60, 61 Although the strong correlation between H3K4 methylation and active transcription is well documented, how SET1/MLL methyltransferase complexes are recruited to specific gene loci is still an open question. However, it has been shown that SET1/MLL complex subunits HCF-1 (host cell factor-1) and menin are required for proper recruitment of these complexes to herpesvirus immediate early promoters and HOX genes, respectively.62, 63 In addition, cell-type-specific transcription factors and cofactors were also shown to mediate recruitment of H3K4 methyltransferases. For example, the transcription factors bZIP28 and bZIP60 bring SET1/MLL complexes to endoplasmic reticulum stress-responsive genes through interactions with Ash2 and WDR5a.64 A direct interaction with p53 was shown to be responsible for recruitment of the SET1 complex to DNA damage-responsive genes.65 In addition, it was reported that the Paf1 transcription elongation complex mediates interactions between Set1 and the C-terminal domain of RNA polymerase Authoring Related - Crack Key For U such that the Set1 complex can be recruited to transcribing genes.66

Transcriptional coactivators that recognize H3K4 methylation

Each state of H3K4 methylation recruits distinct downstream effectors containing specific ‘reader’ domains that further regulate gene expression. Several chromatin remodelers are known to read H3K4 methylation and participate in the regulation of gene expression. For example, the ATP-dependent chromatin-remodeling enzyme, CHD1, recognizes H3K4me2 and H3K4me3 through its two N-terminal chromodomains.67 In addition, BPTF (bromodomain PHD finger transcription factor), a subunit of the ATP-dependent chromatin remodeling complex NURF (nucleosome remodeling factor), was shown to interact with H3K4me3 via its PHD domain.68 As an example of a general transcription factor that binds to H3K4 methylation, transcription factor IID was shown to be recruited to H3K4me3 through its PHD domain-containing TAF3 subunit, resulting in more efficient preinitiation complex formation.69, 70

The activity of several histone-modifying enzymes is modulated by recognition of H3K4 methylation. For instance, the yeast SAGA complex binds to H3K4me2 or H3K4me3 through its Sgf29 subunit, which contains a C-terminal H3K4me2- or H3K4me3-binding tudor domain, and thus efficiently acetylates neighboring histones.71 The acetyltransferase activity of the NuA3 histone acetyltransferase complex can be efficiently targeted to H3K14 through recognition of H3K4me3 by the Yng1 subunit, which contains a PHD domain.72 In addition, the HBO1 histone acetyltransferase complex was reported to acetylate histone H3 in a manner that depends on the PHD domain-containing ING4 (inhibitor of growth family member 4) subunit and facilitates apoptosis by enhancing the expression of genotoxic stress-responsive genes.73

In addition, H3K4 methyl-binding domains within H3K4 methyltransferase complexes further contribute to the precise regulation of their enzymatic activities. For example, the PHD domain-dependent binding of CFP1 to H3K4me3 is responsible for recruitment of SET1A/B complexes to H3K4me3-containing chromatin regions.74 CFP1 was also found to be required for deposition of H3K4me3 near the promoters of DNA damage-responsive genes in ESCs.75 Among MLL family H3K4 methyltransferases, only MLL1 is recruited through its PHD domain to regulate HOX gene expression. The roles of PHD domains within other MLL family proteins remain to be characterized.76, 77

H3K4 methylation and cancers

Mutations in H3K4 methyltransferases highly increase the susceptibility to various cancers.78 About 70% of infant leukemia is related to chromosomal translocation of MLL1 genes, which results in fusion of its N-terminal fragment to more than 50 partner proteins. MLL1 powerful Video Downloader supports downloading - Free Activators are also frequently found in mixed-lineage leukemia, acute lymphoblastic leukemia and acute myeloid leukemia.79 Interestingly, aberrant H3K4 methylation caused by MLL1-AF9 fusion proteins was shown to require an intact MLL1 protein.80 Therefore, considerable effort has been devoted to developing inhibitors that target MLL1 as a cancer therapy strategy. Although inhibitors that directly target the MLL1 SET domain have not yet been discovered, the Authoring Related - Crack Key For U compounds MM102 and MM-401, which specifically disrupt the interaction between WDR5 and MLL1, but not other SET1/MLL family methyltransferases, has been reported to inhibit proliferation of leukemia cells.81, 82

Mutations in H3K4 demethylases are also related to a number of diseases. JARID1 family proteins often function as transcriptional corepressors that are important for expression of development-related genes. JARID1A was shown to be important in regulating HOX gene expression in Caenorhabditis elegans.52 It has also been reported that cryptic fusion of NUP98 (nucleoporin 98) and JARID1A causes HOXA/B gene overexpression and results in pediatric acute leukemia in humans.83 Increased expression of JARID1B, often found in breast carcinomas and testicular cancer, causes misregulation of 14-3-3σ, BRCA1 (breast cancer 1, early onset), CAV1 (caveolin 1) and HOXA5 (homeobox A5) genes.53 X-linked mental retardation patients have a large number of sense or missense mutations in JARID1C genes, implying an important role for the encoded protein during brain development.84 In addition, a number of inhibitors have been developed for LSD1, which is involved in embryonic development and hematopoiesis, and many types of cancer. Some of these inhibitors were designed to irreversibly deactivate LSD1 by forming a covalent adduct with the cofactor FAD within LSD1.85

H3K4 and H3K27 methylations and bivalent domains in ESCs

Bivalent chromatin has an important role in regulating changes in gene expression from poised to active or inactive states in ESCs.15 In bivalent promoters, coenrichment of active H3K4me3 and repressive H3K27me3 marks was shown be responsible for differentiation into specific cell types.61, 86 It was further found that MLL2 is mainly responsible for H3K4 methylation on bivalent promoters.87, 88 H3K27 methylation is an abundant modification that is crucial for fate determination in ESCs. In embryonic fibroblast cells, H3K27me3 was shown to be highly enriched at the promoters of thousands of genes that are responsible for embryonic development and differentiation.89 Subunits of the H3K4 methyltransferase complex also participate in ESC fate determination. For example, WDR5 interacts with Oct4 (octamer-binding transcription factor 4) and mediates H3K4 methylation at key development loci in ESCs.90 During ESC differentiation, ASH2L downregulation correlates with decreased expression of pluripotent transcription factors and increased expression of differentiation-related genes.91 In addition, a specific role for DPY30 in the differentiation of ESCs, but not the maintenance of coreldraw x7 serial number self-renewal capacity, has also been reported.92

H3K9 methylation

H3K9 methyltransferases

H3K9 methylation is a histone modification that is a well-known indicator of silenced transcription and heterochromatin structure.86 Fission yeast has a single H3K9 methyltransferase (Clr4/KMT1) that is responsible for all three states of H3K9 methylation93 and regulates silencing at pericentromere and mating-type loci.94, 95 In mammalian cells, several H3K9 methyltransferases—SUV39H1/KMT1A, SUV39H2/KMT1B, SETDB1/KMT1E, dimeric G9a/KMT1C-GLP (G9a-like protein)/KMT1D and PRDM family—with different catalytic activities and target genes, have roles in diverse cellular events.96 SUV39H1 and SUV39H2 catalyze H3K9 di- and trimethylation in constitutive heterochromatin, including the pericentromeric region.7, 97 Recombinant SUV39H1/2 proteins were shown to possess H3K9 mono- di- and trimethylation activity;98 however, a cell line lacking SUV39H1/2 proteins was shown to lose H3K9me2 and H3K9me3, but not H3K9me1, marks.99 SETDB1 catalyzes H3K9 monomethylation at the pericentromeric region and provides a substrate for SUV39H1/2 to produce H3K9me3.100 Another H3K9 Authoring Related - Crack Key For U, a heterodimer of G9a and GLP (G9a-GLP), mono- and Authoring Related - Crack Key For U H3K9 in euchromatin regions to repress gene expression.101 When either G9a or GLP is deleted, H3K9me1 and H3K9me2 levels are reduced in euchromatin. Interestingly, in vitro analyses have shown that G9a and GLP can individually form homodimers that exhibit H3K9 mono- di- and trimethylation activity.98, 102 In a related observation, the multi-zinc-finger-containing protein Wiz interacts with the G9a-GLP heterodimer and stabilizes its conformation,103 indicating that Wiz-assisted G9a-GLP Authoring Related - Crack Key For U formation is crucial for modulation of G9a-GLP enzymatic activity in vivo.104 Several PRDM (PRDI-BF1 and RIZ homology domain) proteins also contribute to H3K9 methylation. Among 17 members of the PRDM family (PRDM1–17), all of which contain a PR domain similar to the SET domain,105 several are known to possess intrinsic H3K9 methyltransferase activity, whereas the remaining members regulate H3K9 methylation by interacting with other H3K9 methyltransferases, such as G9a.106 However, whether PRDM protein-mediated regulation of H3K9 methylation is direct or indirect is still controversial, and more detailed biochemical studies are necessary to clarify this issue.

H3K9 demethylases

Three classes of mammalian proteins—JHDM2/KDM3, JHDM3(JMJD2)/KDM4 and PHF8/KDM7—have H3K9 demethylation activity. Three JHDM2 (jumonji domain-containing histone demethylase-2) family proteins (JHDM2A–C) have the ability to demethylate H3K9me1 and H3K9me2107 and regulate hormone-dependent transcriptional proshow gold 9.0 3797 registration key - Crack Key For U JHDM3 family proteins can demethylate H3K9me2 and H3K9me3 in addition to H3K36me2 and H3K36me3 in vitro.107, 108, 109 PHF8, a member of the PHF (PHD finger) protein family that acts as a demethylase for H3K9me1 and H3K9me2,110 is a mononuclear Fe(II)-dependent hydroxylase that uses 2-oxoglutarate and oxygen as cosubstrates.111 Because PHF8, like other members of the PHF family, contains a PHD domain, it preferentially removes H3K9 methylations from H3K4me3-containing histone peptide substrates.112 This activity would largely account for the mutually exclusive distribution of H3K4 and H3K9 methylations.

Cross-talk between H3K9 methylation and DNA methylation

A number of studies have reported physical and functional interactions between H3K9 methyltransferases and DNA methyltransferases (DNMTs). For example, SUV39H1/2 and DNMT3A/B interact which each other and can be recruited via their interaction with HP1 (heterochromatin protein 1) to methylate H3K9-enriched constitutive heterochromatin regions, thereby reinforcing the condensed chromatin structure.113, 114 In addition, DNMT3A/B interacts with G9a-GLP and is involved in facultative heterochromatin formation in ESCs.115, 116 Moreover, the fact that G9a, DNMT1 and PCNA (proliferating cell nuclear antigen) are colocalized at the replication fork117 implicates H3K9 methylation in the maintenance of DNA methylation during DNA replication. UHRF1 (ubiquitin-like, containing PHD and ring finger domain 1) also has been reported to have a role in maintaining DNA methylation by bringing DNMT1 to the replication fork through its interaction with methylated H3K9, hemimethylated CpG and DNMT1.117, 118 Furthermore, the methyl-CpG-binding protein MBD1 was shown to recruit SETDB1 to the chromatin assembly complex CAF-1, facilitating SETDB1-mediated methylation of H3K9 on newly deposited nucleosomes.119

Establishment of pericentromeric heterochromatin

SETDB1 and SUV39H1/2 are recruited to pericentromeric heterochromatin and catalyze H3K9 methylation.7, 100, 120 HP1α and HP1β bind to H3K9me3 through their chromodomains and form multimers that interact with SUV39H1/2. As SUV39H1/2 also contains a chromodomain, they can be further recruited to methylated H3K9 at the pericentromeric region by HP1α/β as well as by themselves.97, 121 These multiple interactions enable SUV39H1/2 to spread H3K9me3 to neighboring nucleosomes.122 HP1α/β contributes to heterochromatin formation by recruiting many other proteins involved in heterochromatin formation, such as histone deacetylase, transcriptional repressors and chromatin remodelers.123, 124, 125 H3K9 methylation also increases nucleosome occupancy and has an important role in maintaining transposon repression in pericentromeric heterochromatin.126 In fission yeast and plants, heterochromatin formation mediated by interactions of methylated H3K9 with the RNA interference machinery has been well established.127, 128

H3K9 methylation-mediated transcriptional repression

The G9a-GLP heterodimer deposits H3K9me1 and H3K9me2 and represses target gene expression.101 G9a-GLP can be recruited to target gene promoters through direct interactions with diverse DNA-binding proteins.103, 129, 130, 131 Once it binds and methylates H3K9 on target genes, G9a-GLP recruits additional dimers through its ankyrin repeat domain and spreads H3K9me1 and H3K9me2 to neighboring nucleosomes.101 The resulting repression of gene expression is crucial in many biological processes, such as memory formation, immune responses and differentiation.132, 133, 134 During ESC differentiation, G9a/GLP-mediated facultative heterochromatin formation silences Oct3/4 and Nanog.101 Furthermore, G9a-GLP regulates H3K9me2 and represses different sets of genes in a tissue-specific manner. G9a/GLP-mediated silencing also represses non-neuronal genes in neurons135 and skeletal muscle genes in brown adipose tissue.136 In the immune system, H3K9me2 inhibits uncontrolled interferon induction and regulates naïve T-helper cell differentiation.133 Moreover, H3K9me2 is dynamically altered in the hippocampus and entorhinal wps office login in response to contextual fear conditioning, and mediates memory formation.132 These observations indicate that the distribution of H3K9me2 in euchromatin is dynamically altered according to cell type and external stimuli. Collectively, the results of these studies imply that G9a-GLP interacts with a large number of DNA-binding proteins in different contexts. Numerous studies have revealed that diverse biological processes are regulated by H3K9 methylation. However, only a few downstream target genes, and no upstream regulators of H3K9 methyltransferases, have been identified. Clarifying the detailed molecular mechanisms by which H3K9 methyltransferases mediate transcriptional repression will require a greater effort to identify upstream regulators that bring H3K9 methyltransferases to target genes.

Interestingly, it has been reported that H3K9me3 and HP1γ are enriched in the coding region of certain active genes in several cell lines.137 In contrast to HP1α/β, HP1γ recruits elongating RNA polymerase II and induces gene expression.137 Consistent with Authoring Related - Crack Key For U, the D. melanogaster HP1γ homolog, HP1c, recruits the histone chaperone FACT to RNA polymerase II and enhances transcription elongation of heat-shock genes.138

H3K9 methylation and alternative splicing

Alternative splicing is regulated by nucleosome occupancy and post-translational modifications in transcribing genes.139, 140 A recent study has revealed that local increases in H3K9me2 and H3K9me3 enhance exon inclusion, whereas H3K9 demethylation correlates with exon skipping. Moreover, a genome-wide study showed that H3K9me2 and H3K9me3 are enriched in internal exons.141 Although how H3K9 methylation facilitates exon inclusion is not fully understood, two hypotheses have been proposed. First, H3K9 methylation-bound Dr. fone reviews could recruit the splicing regulatory protein SRSF1 for efficient splicing.142 Alternatively, H3K9 methylation would increase nucleosome occupancy and slow down RNA polymerase II elongation, prolonging the time for RNA splicing.143, 144

H3K9 methylation-related diseases

H3K9 methylation is often misregulated in various diseases, such as neurodegenerative diseases, drug addiction and cancer.145, 146 In a mouse Alzheimer disease model, aberrantly increased H3K9 methylation levels in the BDNF (brain-derived neurotrophic factor) gene leads to downregulation of BDNF expression in neurons. BDNF is critical for synaptic plasticity; hence, reduced BDNF levels are thought to be an important contributor to the pathogenesis of Alzheimer disease.147 In addition, overexpression of SETDB1 and elevated levels of H3K9 methylation are often found in Huntington disease patients.148 Although a correlation between abnormal H3K9 methylation and neurodegenerative diseases is well established, more studies are required to identify target genes through which misregulated H3K9 methylation causes these diseases.

Uncontrolled H3K9 methylation is also linked to cancer. In particular, G9a is often overexpressed in various types of cancer.149, 150, 151 Overexpression of G9a hypermethylates H3K9 on tumor suppressor genes and thus represses their expression. The tumor suppressors, DSC3 (desmocollin 3) and MASPIN (mammary serine protease inhibitor) in breast cancer152 and CDH1 (cadherin 1), DUSP5 (dual specificity phosphatase 5) and SPRY4 (sprouty homolog 4) in ovarian cancer,149 are often silenced by G9a. These results suggest that H3K9 methyltransferases could be good therapeutic targets in cancer treatment.

H3K27 methylation

H3K27 methyltransferases

H3K27me3 is a hallmark of transcriptional repression. The EZH2 (enhancer-of-zest homolog 2) subunit (also known as KMT6A) within PRC2 (polycomb repressive complex 2) complex, an evolutionarily conserved class of polycomb group proteins, is an H3K27 methyltransferase153 responsible for all three states of H3K27 methylation.154, 155, 156 The mammalian PRC2 complex is composed of four core subunits: EZH1/2, SUZ12, EED and RbAp46/48.157, 158 PRC2 can also associate with other accessory proteins, such as AEBP2 (AE binding protein 2), JARID2 and PCLs (polycomb-like proteins).159 These accessory proteins are thought to be involved in recruiting PRC2 to target genes and regulating its activity.158 EZH1/2 alone has no enzyme activity, but incorporation into a PRC2 complex with other subunits enables it to methylate H3K27.160, 161 The PRC2 subunits SUZ12 and EED have the ability to bind the histone H3 N-terminal tail ESET Internet Security Crack - Crack Key For U H3K27me3, respectively. Thus, a positive feedback mechanism is used to spread H3K27me3-repressive marks to adjacent gene loci.162, 163, 164

H3K27 demethylases

UTX/KDM6A, UTY/KDM6C and JMJD3/KDM6B demethylate H3K27me2 and H3K27me3 and are primarily involved in gene derepression.165, 166, 167 UTX contains six TPR (tetratricopeptide repeat) domains and one JmjC domain. The evolutionarily conserved TPR domain mediates multisubunit complex assembly.165 UTX, one of the subunits in the MLL4 H3K4 methyltransferase complex, mediates crosstalk between H3K4 and H3K27 methylations.168 Interestingly, UTX target genes responsible for cancer proliferation and invasiveness are also regulated by MLL4, as evidenced by the fact that individual knockdown of UTX or MLL4 phenocopies the traits of breast cancer cells.169 JMJD3, a JmjC-domain-containing protein that catalyzes demethylation of H3K27me2 and H3K27me3,165 activates transcription of development-related genes and directs differentiation of ESCs into definitive endoderm.170 Microbial stimuli cause nuclear factor-κB-mediated enrichment of JMJD3 at the transcription start site of lipopolysaccharide-responsive genes in macrophages.171 PHF subfamily proteins, including KIAA1718/KDM7A and PHF8, also demethylate H3K27 using α-ketoglutarate and iron as cofactors. KIAA1718 contains PHD and JmjC domains, which allow selective demethylation of H3K27me2 on H3K4-trimethylated nucleosomes.112 The JmjC domain-containing PHF8 is specific for H3K27me2.110

Maintenance of gene repression

The PRC2 subunit EED also binds to H3K27me3 through its WD40 domain, and disruption of this interaction leads to reduced H3K27 methylation and developmental defects.162 Binding of PRC2 to H3K27me3 spreads H3K27 methylation to neighboring nucleosomes, and thus has and important role in the maintenance of gene-expression status. Consistent with a repressive role of H3K27 methylation, the inactivated X chromosome in mammalian cells is highly enriched for H3K27 methylation, which stabilizes the inactive chromatin structure.172

H3K27 methylation and cancers

Misregulation of H3K27 methylation is associated with tumorigenesis as well as metastasis. In this context, overexpression of the PRC2 subunit EZH2 has been reported in human breast and prostate cancers as well as lymphoma.173, 174, 175, 176 For instance, an Y641F mutation in the EZH2 SET domain increases H3K27me3 levels and contributes to the pathogenesis of germinal center B-cell lymphomas.177 In addition, another EZH2 mutant lymphoma cell line harboring an A677G mutation shows elevated levels of H3K27me3 and decreased levels of H3K27me2 and H3K27me1. A structural study reported that the A677G mutation enlarges the lysine tunnel, enhancing the ability of PRC2 to catalyze H3K27 dimethylation.178 These results suggest that inhibition of hyperactive PRC2 could be a potential treatment strategy for specific types of lymphoma. In this regard, several EZH2 inhibitors that target B-cell and follicular lymphomas have been developed.17 For example, the EZH2 inhibitor GSK126 was shown to decrease global levels of H3K27 methylation, reactivate PCR2 target genes, and decrease tumor progression in a mouse model.179

H3K27 methylation can be recognized by chromodomain- and WD40 domain-containing proteins. Among several chromodomain-containing proteins that bind to H3K27 methylation, the PRC1 complex subunit CBX7 (chromobox 7) has received attention as a therapeutic target owing to its involvement in tumorigenesis as well as stem cell self-renewal and differentiation. The recently developed chemical compound, MS37452, binds the methyl-binding pocket of the chromodomain of CBX7, resulting in transcriptional derepression of PRC1 complex target genes and inhibition of the proliferation of prostate cancers.180

H3K36 methylation

H3K36 methyltransferases

In yeast, a single H3K36 methyltransferase, Set2/KMT3, catalyzes all three states of H3K36 methylation.181 The SRI (Set2 Rpb1 interacting) domain in Set2 enables it to interact with the S2- and S5-phosphorylated C-terminal domain of RNA polymerase II and methylate H3K36 during transcriptional elongation.181 Mammalian cells contain at least eight H3K36 methyltransferases: NSD1/KMT3B, NSD2/KMT3G, NSD3/KMT3F, SETD2/KMT3A, SETD3, SETMAR, SMYD2/KMT3C and ASH1L/KMT2H.182 Among these, NSD1–3 and SETD2 are considered major H3K36 methyltransferases. Only SETD2 can catalyze H3K36 trimethylation, whereas the methyltransferase activity of the other seven enzymes is restricted to H3K36 mono- and/or dimethylation.183 NSD (nuclear receptor-binding SET domain) enzymes have additional methylation sites on histones as well as non-histone target proteins.184 Biochemical analyses have shown that NSD enzymes lose their H3K36 specificity if histone octamers are used as a substrate instead of physiologically relevant nucleosomes.184 From a structural perspective, NSD1–3 enzymes have an autoinhibitory loop that blocks the substrate-binding site. Interaction of a short segment of nucleosomal DNA appears to interact with the autoinhibitory loop, dislodging it from the substrate-binding site. Therefore, H3K36 located close to the nucleosome core region can enter the substrate-binding site, thus imparting H3K36 specificity on NSD enzymes.185 The H3K36 trimethyltransferase SETD2 has important roles in many biological processes.186, 187, 188, 189 Although recombinant SETD2 was shown to generate all three states of H3K36 methylation in vitro, only H3K36me3 levels were found to decrease in SETD2-knockdown cells.186, 187 In addition, it was reported that not only H3K36me1 and H3K36me2 levels but also H3K36me3 levels are decreased in NSD1–3-deficient cells.190 These results suggest that NSD enzymes provide H3K36me1 and H3K36me2 to SETD2, which then subsequently generates H3K36me3 in vivo.

H3K36 demethylases

There are two H3K36 demethylase families in mammalian cells: JHDM1/KDM2A-B and JHDM3/JMJD2/KDM4A-D. The H3K36me1- and H3K36me2-specific demethylase, JHDM1, contains multiple histone-binding domains, including a PHD domain,10, 191 and it accounts for mutually exclusive distribution of H3K4me3 and H3K36me3 in same genes.60, 192 JHDM3 is specific for H3K36me2 and H3K36me3 demethylation and has also been shown to demethylate H3K9me2 and H3K9me3 in vitro.109, 111

Regulation of transcription initiation and elongation

H3K36 methylation has been implicated in preventing abortive initiation of transcription within the gene body and in regulation of transcription elongation. In yeast, Set2 binds to the phosphorylated C-terminal domain of RNA polymerase II and catalyzes H3K36 methylation in newly deposited nucleosomes.182 The RPD3 deacetylase complex is recruited to nucleosomes through recognition of H3K36me1 and H3K36me2, and then deacetylates histones. Local deacetylation maintains a repressive chromatin state that prevents aberrant transcription initiation.193 In mammalian cells, different players participate in this process. NSD3, LSD2 (lysine demethylase 2) and G9a form a complex that interacts with transcription-elongation factors and the S2-phosphorylated C-terminal domain of RNA polymerase II. This complex, in turn, maintains newly incorporated nucleosomes in a repressed state by methylating H3K9 and H3K36, and demethylating H3K4.194 Moreover, PWWP domain-containing NSD2 and DNMT3A bind to H3K36-methylated nucleosomes and further contribute to repression of aberrant transcription.194, 195 Collectively, these observations indicate that H3K36 methylation, H3K9 methylation and DNA methylation simultaneously accumulate in newly incorporated nucleosomes to control accurate transcription elongation in mammalian cells.

In addition to its preferential localization to the gene body, H3K36 methylation is also enriched in the promoter region of several genes.196 Promoter-enriched H3K36 methylation inhibits activity of the PRC2 complex and prevents PRC2-mediated expansion of H3K27 methylation.197 This observation is further supported by chromatin immunoprecipitation sequencing analyses showing a mutually exclusive distribution H3K27 methylation and H3K36 methylation.198

DNA damage responses

Several studies have demonstrated roles of H3K36 methylation in DNA damage repair. SETD2 induces DNA mismatch repair by catalyzing H3K36me3 at mismatch sites.199 In addition, the H3K36 dimethyltransferase, SETMAR, facilitates non-homologous end joining at DNA double-strand break (DSB) sites.200 When a DSB occurs, SETMAR is recruited to DSB sites and catalyzes H3K36 methylation. NBS1, a subunit of the MRN complex, and Ku70 recognize H3K36 methylation and stabilize DSB until other non-homologous end-joining -related proteins are recruited. Homologous recombination is also known to be regulated by H3K36me3.201 If a DSB occurs, the H3K36me3-interacting protein LEDGF/p75, also known as PSIP1 (PC4- and SFRS1-interacting protein 1), recruits CtIP (C-terminal-binding protein-interacting protein), which carries out DSB resection. RPA (replication protein A) and RAD51 are then recruited to the site and promote homologous recombination repair.201 In the absence of SETD2, homologous recombination repair cannot be performed properly because reduced H3K36me3 levels lead to dissociation of LEDGF from chromatin. This explains why SETD2 functions as a tumor suppressor. Homologous recombination repair is a very accurate DNA-repair process, and it is usually carried out in H3K36me3-enriched coding regions.183 Therefore, H3K36me3 seems to function as surveillance for DNA damage in coding regions to maintain genome stability.

H3K36 methylation and exon exclusion

Similar to H3K9 methylation, H3K36 methylation also has a role in alternative splicing.139 For example, SETD2-mediated H3K36 trimethylation stimulates exon exclusion.202 H3K36me3-bound MORF4L1 (mortality factor 4-like 1; also known as MRG15) recruits PTB (polypyrimidine tract binding protein), which is a well-known exon-inclusion repressor.139 In addition, a recent study showed that H3.3K36me3-recognizing ZMYND11 protein causes large-scale intron retention.203 These studies imply that H3K36me3 exclusively marks exons in the alternative splicing process. In support of this, H3K36me3 levels are very low in intron-less genes.204 Alternative splicing is also regulated by the level of SETD2.189 Specifically, downregulation of SETD2 levels by polyubiquitylation mediated by the E3 ubiquitin ligase complex, SPOP/CUL3, reduces H3K36me3 on SETD2 target genes and induces their splicing alternatively. Taken together with studies on H3K9 methylation, these results indicate that various histone modifications participate actively in RNA splicing processes.

H3K36 methylation-related diseases

Misregulation of H3K36 methylation often leads to various diseases.182 A defect in NSD1 was reported to cause Sotos syndrome, a neurological disorder characterized by macrocephaly and cognitive and motor skill deficiencies.205 In addition, Ndh2-knockout mice die shortly after birth with symptoms of Wolf-Hirschhorn syndrome.186 These studies suggest that a deficiency of H3K36 methyltransferases disrupts neuronal development, although additional investigation will be required to elucidate the detailed molecular mechanism. The NSD genes also often function as oncogenes when overexpressed or translocated to other genes.206, 207 For instance, a fusion protein caused by joining of the NSD1 gene with the NUP98 gene causes acute myeloid leukemia.208 This NSD1–NUP98 fusion protein facilitates H3K36 methylation and induces inappropriate activation of HOX genes.206 In addition, NSD2 overexpression induces multiple myelomas by promoting the expression of several oncogenes, such as TGFA (transforming growth factor alpha), MET and p21.207 Thus, their causal role in such distinctive diseases strongly suggests that NSD1 and NSD2 have different downstream target genes, despite sharing a common methylation site on histones. Moreover, the fact that a loss-of-function mutation of SETD2 causes renal cell carcinoma development implies that SETD2 acts as a tumor suppressor209, 210 reflecting its roles in DNA repair and alternative splicing.

H3K79 methylation

H3K79 methyltransferases

Unlike other histone lysine methylations, which are located on unstructured histone tail domains and are catalyzed by SET domain-containing methyltransferases, H3K79 methylation occurs on the globular domain of histone H3 and is mediated by Dot1, which lacks a SET domain.211 Dot1 was originally identified as a disruptor of telomeric-silencing genes in yeast,212 and subsequent genetic and biochemical studies showed that Dot1, as well as evolutionarily conserved homologs, including human DOT1L (DOT1-like)/KMT4, are responsible for all three states of H3K79 methylation.213, 214, 215, 216, 217

On the basis of structural studies, Dot1 homologs are categorized as a class I S-adenosyl methionine-dependent methyltransferase.218, 219 Despite structural similarities between human DOT1L and the arginine methyltransferase PRMT1,211 Dot1 homologs exhibit methyltransferase specificity toward lysine rather than arginine.213, 214, 220 In this context, it will be interesting to determine whether Dot1 homologs methylate arginine rather than lysine residues, if present, on novel target proteins.

Intriguingly, in vitro histone methyltransferase assays have demonstrated that Dot1 proteins preferentially methylate nucleosomal substrates rather than free histone H3.214, 220, 221 In addition, it has been shown that a positively charged region within human DOT1L (amino-acid residues 390–407) is required for direct interaction with nucleosomes and histone methyltransferase activity.218 These observations suggest that a newly created surface on the nucleosome produced by participation of histones and DNA provides an environment preferential for recognition and methylation by Dot1 proteins.

Another interesting feature of H3K79 methylation, like that of H3K4 methylation, is the trans-tail histone modification relationship with H2B ubiquitylation. Yeast genetic studies have demonstrated that defects in H2B ubiquitylation cause the complete disappearance of H3K79me2 and H3K79me3.39, 222, 223 Importantly, biochemical approaches have further shown that H2B-ubiquitylated recombinant nucleosomes serve as a preferential substrate for human DOT1L,217, 224 indicating that H2B ubiquitylation directly stimulates the H3K79 methylation activity of human DOT1L. The Muir group has made a number of important observations that have helped elucidate the molecular mechanism underlying this interesting trans-tail histone modification. An enzyme kinetic analysis suggested that human DOT1L undergoes a conformational change in the presence of H2B-ubiquitylated nucleosomes.217 In addition, mutation analyses of the surface of ubiquitin linked to nucleosomes have suggested communication of specific amino-acid residues on ubiquitin with human DOT1L.225 Furthermore, a targeted photocrosslinking study provided evidence that H2B ubiquitylation ‘corrals’ human DOT1L into an H3K79-proximal orientation.226 Collectively, these studies support the proposition that installation of ubiquitin on nucleosomes converts dominant, but unproductive, interactions between DOT1L and the nucleosome into less dominant, but productive, interactions that allow H3K79 methylation.

In addition to H2B ubiquitylation, another trans-tail mechanism for regulating H3K79 methylation has been reported. An in vitro analysis showed that a direct physical interaction between Dot1 and the histone H4 N-terminal tail is required for Dot1-mediated H3K79 methylation.227 In a related observation, increased H4K16 acetylation induced by SAS2 (something about silencing 2) overexpression was shown to cause upregulation of H3K79 methylation by inhibiting the interaction between Sir3 (silent information regulator 3) and the histone H4 tail.227 Collectively, these observations suggest that, not only are structural features of the nucleosome important, surrounding histone modifications also affect the overall H3K79-methylation activity of Dot1.

Unidentified H3K79 demethylase

Although many demethylating enzymes responsible for histone lysine methylations have been reported, the reversibility of H3K79 methylations has not been clarified. The fact that H3K79me2 is reduced to a lesser extent on non-replicating extrachromosomal DNA than on chromosomal loci suggests that removal of H3K79 methylation merely depends on replication-dependent histone exchange.228 However, several studies have shown that global H3K79 methylation levels change dynamically during the G1/S transition.

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