Page 48 - Hematology_ Basic Principles and Practice ( PDFDrive )

20 Part I Molecular and Cellular Basis of Hematology

4 (H3K4me1/2) and acetylation of H3 lysine 27 (H3K27ac) are CHROMATIN REMODELERS
marks of active enhancers, and the degree of H3K27ac is broadly
correlated with enhancer activation. H3K27ac is the enhancer mark Chromatin remodeling alters the position, occupancy, or histone
most commonly used to define superenhancers. composition of a nucleosome within chromatin. Adenosine triphos-
Several histone modifications are particularly associated with phate (ATP)-dependent changes in nucleosome position and occu-
repressed genes: trimethylation of H3 lysine 27 (H3K27me3), di- pancy are mediated by the multisubunit chromatin remodeling
and trimethylation of H3 lysine 9 (H3K9me2/3), and trimethylation complexes, which fall into four families: switch/sucrose nonferment-
of H4 lysine 20 (H4K20me3). H3K27me3 is deposited at both able (SWI/SNF), imitation SWI (ISWI), chromodomain helicase
promoters and enhancers by the polycomb repressive complex 2 DNA binding (CHD), and INO80. ATP-independent changes in
(PRC2) and mediates recruitment of PRC1, resulting in chromatin nucleosome position and occupancy can occur in response to tran-
condensation and transcriptional repression. H3K9me2/3 and scription factor binding or through the action of histone chaperones
H4K20me3 are both highly associated with heterochromatin. that can deposit, remove, or exchange histones. Each of these activi-
H3K9me2/3 serves as a binding site for heterochromatin protein 1 ties alters the accessibility of DNA to transcription factors and other
(HP1). HP1 recruits additional histone-modifying enzymes, includ- DNA-binding proteins.
ing the lysine methyltransferases KMT5B and KMT5C that produce Complexes in the SWI/SNF family include the Brg1/Brm-associ-
H4K20me3. ated factor (BAF) complex, polybromo-associated BAF (PBAF)
Stem cells harbor promoters marked by both activating H3K4me3 complex, and Williams syndrome transcription factor including
and repressive H3K27me3. Upon cellular differentiation, these nucleosome assembly complex (WINAC). They contribute to tran-
“bivalent” or “poised” promoters are rapidly converted to either an scriptional regulation and DNA repair. In addition to nucleosome
activated or a repressed state. sliding, SWI/SNF complexes have been implicated in chromatin
The Aurora B kinase phosphorylates histone H3 at serine 10 looping as well as in eviction of H2A/H2B dimers from the nucleo-
(phospho-H3S10), triggering chromosome condensation during some. Members of the INO80 family of complexes participate in
mitosis. Phosphorylation of H2B at serine 14 (phospho-H2BS14) transcription and DNA repair, but they can also catalyze the exchange
mediates chromatin condensation during apoptosis. of histones from the nucleosome structure. For example, SRCAP can
exchange the H2A/H2B histone dimer for a variant H2A.Z/H2B
dimer, which is associated with actively transcribed promoters. The
TRANSCRIPTION FACTORS CHD nucleosome remodeling family is the largest, and its best-
characterized member is the nucleosome remodeling deacetylase
A transcription factor is a protein that binds to specific DNA sequences (NURD) complex. A subset of NURD complexes incorporates the
and contributes to modulation of gene expression. Transcription MBD2 subunit, which preferentially binds methylated DNA, and
factors are the key determinants of the epigenetic state of the cell. promotes the repression of genes through its remodeling and HDAC
They are modular in structure and contain the following domains: activities. Many alternative NURD complexes incorporate different
DNA-binding proteins and can contribute to transcriptional activa-
• A DNA-binding domain (DBD), having high affinity for specific tion. ISWI family chromatin remodeling complexes catalyze the
sequences of DNA sliding of nucleosomes in short increments and participate in nucleo-
• A trans-activating domain (TAD) or trans-repressive domain some spacing after DNA replication, RNA polymerase elongation,
(TRD), mediating protein–protein interactions with transcrip- transcriptional regulation, and DNA damage repair.
tional coregulators Remarkably, cancer genome sequencing studies have identified
• An optional signal-sensing domain (SSD) (e.g., a ligand binding frequent inactivating mutations in chromatin remodelers in a variety
domain), which can modulate DNA-binding and/or protein- of human cancers. The SWI/SNF complex has particularly emerged
binding activity in response to cellular cues as a powerful tumor suppressor whose disruption occurs in nearly
20% of primary human tumors.
DNA sequences having high affinity for transcription factor binding
are often referred to as response elements. Transcription factor binding
to accessible promoters and enhancers recruits additional proteins, EXPERIMENTAL APPROACHES IN EPIGENETICS
such as coactivators/corepressors, chromatin remodelers, histone-
modifying enzymes, and RNA polymerases, to modulate gene As dramatically as high-throughput sequencing has impacted the
expression. ability to understand the genome, its facilitation of epigenomic
Although sequence-specific DNA binding is a defining feature of research has been equally profound. A wide variety of experimental
transcription factors, chromatin accessibility is a key determinant of approaches are in use and in development for epigenomic research,
transcription factor binding. Most transcription factors preferentially but most are predicated on detecting (1) DNA methylation, (2)
bind nucleosome-free DNA. In many cases, a transcription factor protein–DNA interactions, (3) chromatin accessibility, and (4) three-
needs to compete for DNA binding with other transcription factors, dimensional chromatin structure/looping (Fig. 2.3).
histones, and nonhistone chromatin proteins. The competitive A key feature of all of these techniques is the ability to isolate a
balance between nucleosome and transcription factor binding is subset of DNA sequences from the larger genome on the basis of a
critically affected by chromatin remodeling complexes (see later). In specific chromatin feature. This has several practical implications for
practice, only a small fraction of potential response elements is actu- experiments. First, many techniques rely on cross-linking agents such
ally bound, and many experimentally detected transcription factor as formaldehyde to covalently link proteins to each other and to the
binding sites (TFBS) lack canonical response elements. The genome- DNA they bind. Cross-linking rapidly kills cells and “freezes” chro-
wide pattern of transcription factor binding can be determined matin. Second, all of these experimental techniques involve fragment-
experimentally using chromatin immunoprecipitation (ChIP) and ing chromosomes into much smaller pieces, either by physical
next-generation sequencing (ChIP-Seq; see later) and is known as the disruption (sonication) or by endonuclease treatment. Third, the
transcription factor cistrome. chromatin subset of interest is extracted and enriched by immuno-
Different cell types typically express both common and distinct precipitation, isolation of chromatin fragments of specific sizes, and/
transcription factors. Moreover, the cistrome of a transcription factor or sequence-specific amplification via polymerase chain reaction
differs among cell types, reflecting differences in chromatin accessibil- (PCR). Finally, DNA is isolated from this chromatin subset and
ity and helping to define active promoters and enhancers. Master subjected to next-generation sequencing.
transcription factors are a special subset of lineage-defining transcrip- A common technique for determining the genome-wide methy-
tion factors having expression restricted to specific cell types and lome is bisulfite-sequencing. Treatment of DNA with bisulfite con-
demonstrating very high binding at superenhancers. verts cytosine residues to uracil but leaves 5-methylcytosine (5mC)