Question

In your own words, how do chromatin modifications regulate transcription? What modifications are observed in regions of the genome that are being actively transcribed? In regions that are not actively transcribed?

Answer 1

Chromatin structure is highly complex and impressively dynamic. Scientists now realize that the nucleosome, which was once thought to be static, actually plays an integral role in directing some elements of transcriptional specification. A nucleosome core particle consists of eight histone proteins (two each of H2A, H2B, H3 and H4) and 146 base pairs of double-stranded DNA. The composition of nucleosomes is not set in stone, however. Indeed, canonical histones can themselves be replaced by histone variants or modified by specific enzymes, thereby making the surrounding DNA more or less accessible to the transcriptional machinery.
So far, a number of histone variants have been found and localized to specific areas of chromatin. For instance, H2A.Z is a variant of H2A and is often enriched near relatively inactive gene promoters. Interestingly, H2A.Z does not take its place during replication when the chromatin structure is established. Instead, the chromatin remodeling complex SWR1 catalyzes an ATP-dependent exchange of H2A in the nucleosome for H2A.Z (Wu et al., 2005).
CENP-A is another known histone variant that has been found to be associated with centromeres. Originally localized to the centromere through immunofluorescence studies, CENP-A was believed to be involved in centromeric activity during cell division. But, once the CENP-A protein was isolated and sequenced, it was shown to have sequence homology to H3, suggesting that CENP-A actually replaces canonical H3 near the centromere. Some experiments suggest that these variant histones that occur in particular areas of the genome may assist in the specific regulation of chromatin behavior and gene transcription from these areas (Li et al., 2005)
Histone Modification and the Histone Code
Histone sequences are highly conserved. A typical chromatin fiber, with the blue cylinders representing histones. Extending from each of the histones is a "tail," called the N-terminal tail because proteins have two ends--an N terminus and C terminus. Here, the C terminus forms a globular domain that is packaged into the nucleosome. The other end of the histone is more flexible and capable of interacting more directly with DNA and the different proteins within the nucleus. Specifically, histone modification involves covalent bonding of various functional groups to the free nitrogens in the R-groups of lysines in the N-terminal tail. Early research has linked differing levels of acetylation and methylation on the histones to altered rates of DNA transcription (Turner, 2005). While the most common additions are acetylation and methylation of lysine residues, many more types of modifications have also been observed, including phosphorylation, a common posttranslational modification. The different types of modifications, which have been called the "histone code," are put in place by a variety of different enzymes, many of which have yet to be fully characterized. Thus, the story of the remodeling machinery continues to be told through a variety of experiments, and much remains to be revealed.
Nucleosome Positioning and Reorganization
Because eukaryotic DNA is tightly wrapped around nucleosomes and the positive charges of the histones tightly bind the negative charges of the DNA, nucleosomes essentially act as a physical barrier to transcription factors that need to bind to certain regions of DNA. However, specific acetylations can remove the positive charge on the lysine amino group that is acetylated, so the nucleosome "loses its grip" on the DNA. This modification results in a loosening of the coil.
Other remodeling enzyme complexes actually slide the nucleosomes along the DNA to clear them from the promoter regions (Cosgrove et al., 2004). In this case, the remodeling enzymes use the energy from ATP to regulate nucleosome movement. For example, prior to transcription in yeast, one of the major types of chromatin remodeling machines, called the SWI/SNF and SAGA histone acetylase complex, is recruited to the yeast HO gene promoter by the SWI5 activator. Activator-dependent chromatin modification then moves the nucleosome out of the way so that RNA polymerase II can reach the promoter regions of the DNA (Struhl, 1999).
Chromatin remodeling activity by SWI/SNF or other remodeling machines can also be required for recruiting additional chromatin remodeling activity to the site, as well as additional downstream sites. Modifications at a promoter can occur in multiple steps that are independently regulated, and additional modifications can occur stepwise stretching from the point of the first modification along the DNA strand in a downstream direction toward the promoter. These modifications open up an elongated region of active chromatin and allow for a wide range of intermediate, transcriptionally inactive states for the eukaryotic promoter. Promoters can also be poised with RNA polymerase bound but not elongating the mRNA; in yeast, up to 15% of sites have such stalled transcription. Changes in gene expression during the specific developmental stages of an organism or cell coincide with fluctuations in the levels of each of the specific protein complexes involved in chromatin remodeling (Struhl, 1999).

TRANSRIPTION OF NAKED DNA
The principles and mechanisms underlying transcription on naked DNA are remarkably similar between eukaryotes and prokaryotes despite the increased complexity of eukaryotic transcription machinery (Hahn, 2004). The typical RNA polymerase II (Pol II) transcription cycle begins with the binding of activators upstream of the core promoter (including the TATA box and transcription start site). This event leads to the recruitment of the adaptor complexes such as SAGA (Green, 2005) or mediator, both of which in turn facilitate binding of general transcription factors (GTFs; Thomas and Chiang, 2006). Pol II is positioned at the core promoter by a combination of TFIID, TFIIA, and TFIIB to form the closed form of the preinitiation complex (PIC). TFIIH then melts 11–15 bp of DNA in order to position the single-strand template in the Pol II cleft (open complex) to initiate RNA synthesis. The carboxy-terminal domain (CTD) of Pol II is phosphorylated by the TFIIH subunit during the first 30 bp of transcription and loses its contacts with GTFs before it proceeds onto the elongation stage. Meanwhile, the phosphorylated CTD begins to recruit the factors that are important for productive elongation and mRNA processing (Buratowski, 2003).

Histone Modifications and Transcription
Both histone tails and globular domains are subject to a vast array of posttranslational modifications. These modifications include methylation of arginine (R) residues; methylation, acetylation, ubiquitination, ADP-ribosylation, and sumolation of lysines (K); and phosphorylation of serines and threonines. Modifications that are associated with active transcription, such as acetylation of histone 3 and histone 4 (H3 and H4) or di- or trimethylation (me) of H3K4, are commonly referred to as euchromatin modifications. Modifications that are localized to inactive genes or regions, such as H3 K9me and H3 K27me, are often termed heterochromatin modifications. Most modifications are distributed in distinct localized patterns within the upstream region, the core promoter, the 50 end of the open reading frame (ORF) and the 30 end of the ORF. Indeed, the location of a modification is tightly regulated and is crucial for its effect on transcription. For instance, as we will discuss later in more detail, Set2- mediated methylation of histone H3K36 normally occurs within the ORF of actively transcribed genes. However, if Set2 is mistargeted to the promoter region through artificial recruitment, it represses transcription (Landry et al., 2003; Strahl et al., 2002). Typically, histone acetylation occurs at multiple lysine residues and is usually carried out by a variety of histone acetyltransferase complexes (HATs; Brown et al., 2000). Distinct patterns of lysine acetylation on histones have been proposed to specify distinct downstream functions such as the regulation of coexpressed genes (Kurdistani et al., 2004). Another view posits that the biological functions of histone acetylation rely primarily on the number of lysines modified (e.g., a cumulative effect) with the one known exception of H4K16Ac (Dion et al., 2005). In contrast to acetylation, histone methylation, phosphorylation, ubiquitination, etc. are often catalyzed by a specific enzyme at a specific site and result in unique functions. The reason for the distinction between acetylation and other modifications is currently unknown, but biophysical changes caused by histone acetylation may offer a partial explanation. Since their identification decades ago, histone modifications have been proposed to have a number of different functions (reviewed in Workman and Kingston, 1998). However, a consensus has begun to emerge in recent years. First, with the exception of methylation, histone modifications result in a change in the net charge of nucleosomes, which could loosen inter- or intranucleosomal DNA-histone interactions. This idea is supported by the observation that acetylated histones are easier to displace from DNA both in vivo (Reinke and Horz, 2003; Zhao et al., 2005) and in vitro (Chandy et al., 2006; Hassan et al., 2006; Ito et al., 2000). Second, it is well accepted that protein modifications can be recognized by other proteins (reviewed in Seet et al., 2006). Given the diversity of covalent modifications, it has been proposed that individual histone modifications or modification patterns might be read by other proteins that influence chromatin dynamics and function (Jenuwein and Allis, 2001; Strahl and Allis, 2000; Turner, 2000). Therefore, the outcome of a particular modification is dependent on the effector proteins that recognize it. Third, some modifications directly influence higher-order chromatin structure. For instance, acetylation of H4 K16 inhibits the formation of compact 30 nm fibers (Shogren-Knaak et al., 2006). Finally, the mechanisms discussed above are not necessarily mutually exclusive. For example, acetylation of H4 K16 also impairs the efficiency of ATP-dependent chromatin assembly and mononucleosome mobilization by the ACF histone chaperone (Shogren-Knaak et al., 2006), thus suggesting that a single modification can elicit multiple effects on chromatin structure.
Histone Modifications and Transcription
The well-defined landscape of chromatin modifications observed over the body of a gene is striking in its detail. However, as we shall discuss below, it is the consequence of an ordered recruitment of various histone-modifying enzymes. This well-choreographed process is likely a consequence of Pol II moving through the ORF while struggling to maintain chromatin structure within the transcribed region. Histone H3K4 Methylation The H3K4 residue in yeast is methylated by the Set1 complex across the entire ORF of an active gene (reviewed in Shilatifard, 2006).Monomethylation is enriched toward the 30 end, and dimethylation peaks in the middle, whereas trimethylation occurs around the transcription start site and the 50 end of the ORF (Pokholok et al,2005). One possible interpretation of this distribution pattern is that K4me1 occurs at a basal level; Set1 associates with elongating Pol II at the beginning of the ORF and converts monomethyl into dimethyl and eventually into trimethyl. Hence, the gradual addition of methyl groups at the 50 ORF may then lead to the gradient of tri- and dimethyl trailing off at the 30 end of the ORF. This hypothesis is supported by the observation that in a paf1D mutant, both H3K4me2 and H3K4me3 are eliminated, but H3K4me1 is not affected. More importantly, H3K4me1 increases at the 50 ORF (Dehe et al., 2005), presumably due to the failure to convert H3K4me1 into H3K4me2 or into H3K4me3 in the mutant. Therefore, Set1 can catalyze H3K4me1 independent of PAF, but conversion to H3K4me2 or -me3 requires PAF and association with Pol II. This phenomenon resembles how the MLL/WRD5 complex controls H3K4 methylation in humans, where histones can be mono- and dimethylated at H3K4 without WDR5, a WD-40 domain-containing protein (Wysocka et al., 2005). The MLL/WRD5 complex can specifically recognize the dimethyl marks and convert them into trimethylation (Wysocka et al., 2005). This remarkable similarity accentuates the importance of di- and trimethylation of H3K4 in transcription regulation. Coincidently, H2B ubiquitination is only required for di- and trimethylation (Dehe et al., 2005; Shahbazian et al., 2005), and the extent of both modifications is positively correlated to the frequency of transcription. Therefore, it is conceivable that these two marks enriched at the 50 ORF may serve as a critical signal for defining the start of the transcribed domain and the frequency with which Pol II travels through it. However, the precise function of H3K4 methylation is still unknown. In a completely defined in vitro transcription system, it has been shown that H3K4 methylation does not affect transcription elongation per se (Pavri et al., 2006), which is consistent with the in vivo observation that Set1 does not affect elongation or processivity of Pol II (Mason and Struhl, 2005). These data imply that the importance of H3K4 methylation might rest primarily in its signaling functions. Recent studies provide some clues in this direction. Chromatin-remodeling factors (NURF) and histone-modification complexes (hTip60, mSIN3/HDAC, yNuA3, etc.) contain PHD domains that can specifically recognize H3K4 methylation (see review by Zhang, 2006), thereby recruiting their respective complexes to activate/repress transcription. In addition, an elongation-related chromatin-remodeling factor Chd1 also recognizes methylated H3K4 (Pray-Grant et al., 2005; Figure 3A). Future studies addressing how these H3K4-binding complexes influence transcription elongation will be of importance for understanding the role of H3K4 methylation. Histone H2B Monoubiquitination Histone H2B monoubiquitination (H2Bub1) occurs at both promoters and ORFs (Kao et al., 2004; Xiao et al., 2005) and is dependent on PAF and active transcription (Ng et al., 2003a; Pavri et al., 2006; Wood et al., 2003). One commonly accepted role of H2Bub1 in transcription is to stimulate di- and trimethylation of histone H3K4 (Dehe et al., 2005; Shahbazian et al., 2005; Sun and Allis, 2002). Due to the bulky nature of ubiquitin, it has been speculated that its incorporation into nucleosomes would be disruptive to their structure. However, biochemical studies indicate that ubiquitination of histones has very little effect on nucleosome architecture (Jason et al., 2002). Pavri et al. recently reported that monoubiquitination of H2B enhances the rate of transcription elongation on chromatin templates (Pavri et al., 2006). It is noted that in this system the stimulatory effect occurs while H2Bub1 remains in chromatin. This seems in contrast to the in vivo observation that both ubiquitination and deubiquitination are important for full transcription activation at the GAL1 promoter (Henry et al., 2003). However, it is possible that in the in vitro assay, deubiquitination may stimulate transcription to a greater extent than does the initial effect of ubiquitination or that deubiquitination is required only at the promoter. Histone H3K36 Methylation Histone H3K36 methylation mediated by Set2 is another important landmark on chromatin during elongation. Both di- and trimethylation are enriched at the 30 ORF, while only trimethylation displays a positive correlation with transcription rates (Figure 1; Pokholok et al., 2005; Rao et al., 2005). Our understanding of the role of K36 methylation in elongation is much more advanced compared to the role of other modifications in this process. H3K36 methylation is recognized by the chromodomain of Eaf3, a subunit of the Rpd3S histone deacetylase complex. Trimethylation leads to the recruitment of Rpd3S and creates a hypoacetylated environment within ORFs (Carrozza et al., 2005; Joshi and Struhl, 2005; Keogh et al., 2005).
Nucleosomes as Transcription Barriers
The nucleosome forms a strong barrier to Pol II transcription in vitro. Although the phage SP6, T7 RNA polymerases, and yeast Pol III can transcribe through nucleosomal DNA by mobilizing histones along the templates (Clark and Felsenfeld, 1992; Studitsky et al., 1994, 1995, 1997), RNA Pol II can only traverse the nucleosome under conditions in which at least one H2A/H2B dimer is lost (Kireeva et al., 2002, 2005). How does Pol II overcome the nucleosome barrier? When Pol II transcribes into a nucleosomal template, it pauses at certain sites that are presumably related to the strength or nature of the histone-DNA contacts (Bondarenko et al., 2006; Kireeva et al., 2005). This pausing leads to Pol II backtracking. The prototypic transcription elongation factor TFIIS reactivates the backtracked Pol II complexes and promotes transcription through the nucleosomal templates (Kireeva et al., 2005; Kulish and Struhl, 2001). Consistent with this observation, TFIIS was recently found to be a major component of chromatin transcription-enabling activity (CTEA). CTEA strongly stimulates transcription elongation through nucleosomes at a post-PIC step and in a manner dependent upon p300 and acetyl-CoA (Guermah et al., 2006). Biochemical and genetic experiments suggest that the FACT histone chaperone complex can also help Pol II transcribe through nucleosomes (reviewed in Reinberg and Sims, 2006). However, its mechanism is different from that of TFIIS. The requirement for stoichiometric amounts of FACT for nucleosomal transcription initially suggested that FACT might act as a histone chaperone (Orphanides et al., 1999). This notion is consistent with the observation that passage of Pol II through nucleosome at high salt conditions causes a quantitative loss of one H2A/H2B dimer (Kireeva et al., 2002). It was ultimately shown that FACT does act as a chaperone during transcription and that it functions in both disassembly and reassembly of H2A/ H2B dimers (Belotserkovskaya et al., 2003). ATP-dependent chromatin-remodeling complexes have long been suspected of playing a role in helping Pol II pass through nucleosomes (Workman and Kingston, 1998); however, direct evidence did not emerge until recently. Using C-tail DNA templates reconstituted with a histone octamer, Carey et al. demonstrated that RSC can help Pol II transcribe through otherwise paused sites on nucleosome templates. This reaction is further stimulated by SAGA- and NuA4-mediated histone acetylation that presumably utilizes the multiple bromodomains (acetyl-lysine-binding domains) that are contained within the RSC complex (Carey et al., 2006). It will be interesting to see if this effect involves active histone eviction by RSC.

The N terminus.
(A) General chromatin organization. Like other histone "tails," the N terminus of H3 represents a highly conserved domain that is likely to be exposed or extend outwards from the chromatin fiber. A number of distinct post-translational modifications are known to occur at the N terminus f H3 including acetylation (green flag), phosphorylation, and methylation. Other modifications are known and may also occur in the globular domain.
(B) The N terminus of human H3 is shown in single-letter amino-acid code. For comparison, the N termini of human CENP-A, a centromere-specific H3 variant, and human H4, the nucleosomal partner to H3, are shown. Note the regular spacing of acetylatable lysines , and potential phosphorylation and methylation sites. The asterisk indicates the lysine residue in H3 that is known to be targeted for acetylation as well as for methylation; lysine 9 in CENP-A may also be chemically modified.

Signaling Function of Remodeled Chromatin
Histone modification can open chromatin, thus permitting selective binding of transcription factors that, in turn, recruit RNA polymerase II (Turner, 2005). Varying levels and types of histone modifications have been shown to correlate with levels of chromatin activation. For example, one group of researchers used antibody-based immunoprecipitation studies to determine that acetylation of histone H3 and methylation at lysine residue K4 appeared to coincide with each other. They also coincided during transcriptional activation in chicken embryos, while methylation at lysine residue K9 marked inactive chromatin.
DNA Methylation
Another means by which transcription is controlled is through methylation of the DNA strand itself. Not to be confused with histone methylation, methylation of the DNA strand involves cytosine bases of eukaryotic DNA being converted to 5-methylcytosine, resulting in the repression of transcription, particularly in vertebrates and plants. The altered cytosine residues are usually immediately adjacent to a guanine nucleotide, resulting in two methylated cytosine residues set diagonally to one another on opposing DNA strands Heavily methylated regions of DNA with elevated concentrations of these so-called CpG groups are often found near transcription start sites. In an interestingly coordinated process, proteins that bind to methylated DNA also form complexes with proteins involved in deacetylation of histones. Therefore, when the DNA is in a methylated state, nearby histones are deacetylated, resulting in compact, semipermanently silent chromatin. Likewise, demethylated DNA does not draw deacetylating enzymes to the histones, but it often attracts histone acetyltransferases, allowing histones to remain acetylated and promoting transcription.
Summary
Storage of eukaryotic DNA in small, compact nuclei requires that this DNA be tightly coiled and compacted in the form of chromatin. However, the structure of chromatin also appears to serve a second, possibly more important role, in that it gives eukaryotic cells the capability to exert complex levels of control over gene expression.
As described throughout this article, chromatin and the DNA sequences it contains are constantly undergoing modifications, thereby periodically exposing different regions of DNA to transcription factors and RNA polymerases. The cumulative effects of these changes are various states of transcriptional control and the ability of eukaryotic cells to turn genes on and off as needed. This complexity provides eukaryotes with a means of making the most of a relatively small number of genes. However, much research remains to be performed before investigators precisely understand how the many mechanisms of chromatin remodeling operate, as well as how they work together to result in the complex patterns of gene expression characteristic of eukaryotic cells.