Eukaryotic Chromosome Structure and Function

Question 1

What does a nucleosome consist of?

Answer 1

DNA and histone proteins

Question 2

What are the four different histones that make up the octameric protein core of a nucleosome?

Answer 2

H2A, H2B, H3 and H4

Question 3

Describe the general structure of a histone protein.

Answer 3

A three alpha-helix “histone fold” and unstructured TAIL regions

Question 4

The histone protein core is modular - how is this made up?

Answer 4

Two molecules each of H3 and H4 form a tetramer; H2A and H2B molecules form two dimers

Question 5

During regulation of transcription, eukaryotic chromatin-remodelling co-activators/repressors act at two functionally distinct levels. What are these two levels?

Answer 5

1) Local-level remodelling at gene-regulatory regions (promoter/UPE, enhancers etc.)
2) Domain-level remodelling over large chromosomal regions

Question 6

What is the major function of co-activator/repressor complexes at gene-regulatory regions?

Answer 6

To manipulate individual nucleosomes to allow TF and RNA pol II access to DNA

Question 7

What two ways can co-activators and repressors remodel chromatin?

Answer 7

1) Some use energy from ATP hydrolysis to modify nucleosomes
2) Some covalently modify histone protein amino acid residues within nucleosomes

(They are enzymes)

Question 8

What two ways do co-activators and co-repressors manipulate nucleosomes?

Answer 8

1) Nucleosome positioning
2) Nucleosome structure

(Know this because of chromatin-seq/MNase-seq, a modification of DNase-seq)

Question 9

What is the key reagent involved in Chromatin-seq/MNase-seq?

Answer 9

Micrococcal nuclease (MNase)

Question 10

What are the key steps involved in Chromatin-seq/MNase-seq?

Answer 10

1) Living cells + detergent + micrococcal nuclease –> MNase digestion of chromosomes in vivo = fragments of DNA wrapped around a nucleosome protected from digestion
2) The around 150 bp of MNase-resistant DNA fragments from the cells are purified and sequences

Question 11

Chromatin-seq: what does a peak in sequence read frequency tell you?

Answer 11

A peak = positioned nucleosomes. Chromatin-Seq experiments reveal that regions of genome have nucleosomes accurately positioned relative to underlying DNA sequence according to cell type/condition.

Question 12

What is a pioneer TF and give some examples?

Answer 12

A pioneer TF, e.g. Oct3/4 and FoxA, is able to bind nucleosome-associated motifs

Question 13

What is an NFR and when do they form?

Answer 13

Nucleosome-free regions: when a gene is activated, gene-regulatory DNA and promoters become exposed as NFRs to allow access for TF binding

Question 14

How can we map transcription factors?

Answer 14

Using chromatin-seq: MNase will also not cleave DNA when the DNA is bound to TFs, leaving 20-50 bp of undigested DNA which we can sequence

Question 15

What drives the creation of NFRs?

Answer 15

The recruitment of chromatin-remodelling ATPase co-activators, energy from ATP hydrolysis drives non-covalent changes in nucleosome structure

Question 16

What are chromatin-remodelling ATPases?

Answer 16

Complexes that contain a core ATPase sub-unit and other proteins that modulate and target their activity (we have at least 5 classes of chromatin-remodelling ATPase complexes which differ in the structure of the ATPase sub-unit and in the type of nucleosome remodelling events they catalyse)

Question 17

How does the chromatin-remodelling ATPase SWI/SNF act?

Answer 17

1) SWI/SNF ATPases can evict/displace histones from the DNA to remove nucleosomes from underlying sequence
2) They can also SLIDE histone octamers relative to the underlying DNA to alter the position of nucleosomes during gene activation

Question 18

What are histone chaperones?

Answer 18

Histone chaperones (e.g. ASF1) are used to carry histones that are evicted during chromatin remodelling and during nucleosome assembly/dis-assembly during DNA replication/transcript elongation.

Question 19

What effect can SWI/SNF ATPses, such as RSC, have on nucleosome structure?

Answer 19

SWI/SNF ATPases such as RSC can destabilise the overall structure of nucleosomes to make the wrapped DNA more generally accessible to DNA-binding proteins

Question 20

What are two of the main ways to modify nucleosome structure?

Answer 20

1) Incorporating histone variants e.g. H2A.Z; H3.3, H2ABbd
2) Many chromatin remodelling co-activators/repressors (and some TFs) catalyse addition or removal of specific histone PTMs) - amino acids within the histone tails and cores are subject to more than 60 distinct, mostly reversible, PTMs

Question 21

Give an example of an ATPase that can exchange a variant histone into nucleosomes.

Answer 21

SWR/INO80 ATPase can exchange variant histone H2A.Z into nucleosomes

Question 22

Where is the variant histone H2A.Z often found?

Answer 22

Flanking NFR associated with yeast and human promoters - H2A.Z-containing nucleosomes may be more accessible to TFs and RNA pol II

Question 23

What are HAT co-activators, give an example and explain what do they do.

Answer 23

Histone acetyltransferase (HAT) co-activators, e.g. SAGA, add acetyl groups (from acetyl coA) to multiple lysine residues in the N-terminal tails of histones H2B, H3 and H4 to make them more accessible to TF binding and PIC function.

Question 24

What are HDAC co-repressors, give an example and explain what do they do.

Answer 24

Histone deacetylase (HDAC) co-repressors, e.g. RPD3, remove acetyl groups from lysine residues in the N-terminal tails of histones.

Question 25

As well as changing the structure of nucleosomes, histone PTMs also..

Answer 25

…create binding sites for other proteins (often more co-activator/repressor complexes), writing an “epigenetic” code (e.g. bromodomains recognise acetyl-lysines; chromo- and PHD domains recognise methylate lysines)

Question 26

What two histone PTM marks are generally associated with nucleosomes in non-transcribed/”repressed” regions of the genome?

Answer 26

H3K9me2/3 and H4K20me3

Question 27

i) What histone PTM mark is generally associated with non-activated/”off’ state gene regulatory regions and ii) what other active mark is sometimes found with it?

Answer 27

i) H3K27me3.

ii) The “active” PTM H3K4me3 is sometimes found with H3K27me3 at gene-regulatory regions “poised” between “on” and “off”

Question 28

Give some examples of PTMs generally associated with activated/”on” state gene regulatory region nucleosomes.

Answer 28

H3K9ac, H3K27ac and H3K4me2/3

Question 29

How do local chromatin remodelling events at widely-spaced higher eukaryotic gene-regulatory regions communicate?

Answer 29

Activated eukaryotic gene-regulatory regions are often separated in sequence in the nucleus but not in space, they interact with each other

Question 30

How do we know that activated eukaryotic gene-regulatory regions interact with each other?

Answer 30

Chromosome conformation capture (3C) technologies which use NGS to map interactions between DNA regions

Question 31

What are some variants of 3C?

Answer 31

4C, 5C, Hi-C, ChIA-PET

Question 32

What are the key reagents involved in 3C technologies?

Answer 32

1) Formaldehyde
2) Sequence-specific restriction endonuclease
3) DNA ligase

Question 33

What are the key steps of 3C?

Answer 33

1) Cells are treated with formaldehyde to trap local physical interactions between sequences in the nucleus
2) DNA is cleaved using the restriction endonuclease, the interacting enhancer and promoter DNA remains held together by the cross linking
3) DNA is treated with DNA ligase - “proximity ligation”, sequences such as promoters and enhancers that are held in proximity by the formaldehyde become directly joined to each other
4) DNA junctions are purified and sequenced
5) Polt graph of the frequency at which pairs of sequences are found joined to each other - often plotted as a contact probability map

Question 34

What is ChIA-PET and how does it differ from 3C?

Answer 34

Chromatin Interaction Analysis by Paired-End Tag Sequencing. A ChIP step is added to enrich for gene-regulatory sequences using specific co-activators or TFs before DNA junctions are purified and sequenced.

Question 35

What is a contact probability map?

Answer 35

A 3D graph where the length of one region of DNA is plotted on both x- and y-axes, and then the frequency of types of 3C junction sequence reads is plotted as a colour intensity on the x-axis.

Question 36

How do you read a contact probability map?

Answer 36

1) Ignore the signal forming the central diagonal line (these are sequences that are directly adjacent in DNA)
2) Look for signals off the diagonal (the graphs are symmetrical so these always come in pairs)
3) Read horizontally from one axis to hit a signal, then read vertically up to the other axis
4) You only need to look at one side as its a symmetrical image

Question 37

How are gene-regulatory regions held together during gene activation?

Answer 37

1) Gene-specific architectural co-activators e.g. the general co-activator Mediator functions to hold many promoter-enhancer interactions together
2) Looped regions of DNA can be held in contact by cohesins

Question 38

What are cohesins?

Answer 38

Cohesions are ring-like proteins which normally function to hold chromatids together during mitosis and meiosis

Question 39

What is an ACH and how does it form?

Answer 39

An active chromatin hub forms when genes have multiple regulatory regions, TFs at distant enhancers/LCRs and their recruited co-activators/repressors coalesce into ACHs to combine their signalling inputs and activities in one concentrated physical location in the nucleus.

Question 40

What elements are required for strong, well-regulated transcription in all chromosomal locations?

Answer 40

Promoter/UPE, enhancers, and LCR. Promoters do not work on there own; you only get some regulated transcription in some chromosomal locations with just a promoter/UPE and with just an enhancer and promoter/UPE you get strong, well-regulated transcription in some chromosomal locations. The LCR forces the region to become supportive of transcription.

Question 41

How is chromatin organised?

Answer 41

Chains of nucleosomes can fold into various short regions of regular secondary structure however there appears to be no predominant set of hierarchical coiled fibres as previously thought, at the level of individual nucleosomes, both euchromatin and heterochromatin are best modelled as disordered chains with just subtle differences in nucleosome density.

Question 42

What can EM tomography be used for?

Answer 42

Tracing individual nucleosome chains in fixed cells.

Question 43

What does the disordered chain model for chromatin state?

Answer 43

No hierarchy of coiled structures, just different densities of general aggregation. At the scale of individual nucleosomes chromatin is relatively disordered but overall lies a hidden organisation.

Question 44

How are the multiple forms of hetero/euchromatin defined?

Answer 44

By histone PTMs and the factors recruited to them (not by hierarchical compaction)

Question 45

How is heterochromatin re-enforced?

Answer 45

Heterochromatin is self-re-enforced by cycles of co-repressor-mediated histone PTMs. Methylated H3 and H4K resides in silent chromatin domains recruit general heterochromatin repressor complexes such as HP1 and Polycomb factors. Chains of nucleosomes in heterochromatin are drawn together by binding of accessory factors such as HP1 and Polycomb which make inter-nucleosomal links.

Question 46

What is the visible example of HP1-bound heterochromatin?

Answer 46

The pericentromere which forms on either side of centromeres in higher eukaryotes

Question 47

What histone modification does HP1 recognise to form heterochromatin?

Answer 47

H3K9me1-3 (CpG DNA sequences are also methylated in these regions). Both HP1 and a methyl DNA binding protein both recruit H3K9 methylases (histone lysine methylase/KMT) that creates more H3K9me1-3 by adding methyl groups to any local histone tails lacking them, which in turn recruits more HP1 and so on in a spreading positive feedback loop. HP1 therefore helps create the very PTM that directed its own binding to chromatin.

Question 48

How is the conversion of heterochromatin to euchromatin initiated during gene activation?

Answer 48

The TFs that bind to LCRs including pioneers which are specifically able to bind their motifs in inaccessible chromatin.

Question 49

What are LCRs?

Answer 49

LCRs are specialised enhancers that recruit co-activators (histone acetylators) which mediate domain-wide spreading of gene-activating histone PTMs encompassing an entire activated gene and its regulatory regions

Question 50

How is euchromatin spreading induced by LCRs?

Answer 50

Similarly to the process of heterochromatin spreading, TF binding to the LCR recruits chromatin remodelling co-activators and localised chromatin remodelling occurs at the LCR. These co-activators that can bind to their own remodelled product and remodels the next region to induce a wave of euchromatin spreading.

Question 51

How do HATs render gene-regulatory sequences in chromatin more accessible to TFs?

Answer 51

Localised acetylation of histones decreases intra-nucleosomal DNA:histone interactions and inter-nucleosomal interactions at the domain level consequently opening up chromatin domains

Question 52

How are repressive histone methyl PTMs removed?

Answer 52

LCR bound TFs recruit KDM (histone lysine de-methylase) co-activators which remove repressive histone methyl PTMs

Question 53

What enables the formation of ACHs?

Answer 53

Chromatin-remodellers, initially recruited at LCRs, create domains of flexible, open euchromatin

Question 54

What stops euchromatin and heterochromatin states from spreading forever?

Answer 54

Insulators act as boundary elements between chromatin domains and as a barrier to RNA polymerases. They restrict inappropriate interactions between enhancers and promoters.

Question 55

What is the main insulator-binding protein in mammals?

Answer 55

The Zn-finger protein, CTCF (the CCCTC binding factor). It is sometimes a TF but also an architectural factor that tethers insulators (and other CTCF-bound sites) to each other and to intranuclear structures thus physically isolating the LCRs, enhancers and promoter.

Question 56

What defines the extent of flexible ACH regions?

Answer 56

CTCF-bound insulators interact with each other and cohesin, ultimately defining the extent of flexible ACH regions.

Question 57

When genes are activated, not only are regulatory regions remodelled by specific PTMs but so is the coding region, what is the chromatin remodeller?

Answer 57

The TEC (elongating RNA pol II) is a potent chromatin-remodeller in its own right. A variety of chromatin-remodelling co-activator complexes also bind as elongation factors and use covalent and ATP-dependent nucleosome modifying activates to help RNA pol II “bulldoze” through chromatin.

Question 58

What are eRNAs and cryptic promoters?

Answer 58

Enhancer RNAs are a form of long non-coding RNA (lncRNA). Most active gene-regulatory regions recruit RNA pol II to cryptic promoters and initiate eRNA transcripts. Many cryptic promoters (even some mRNA promoters) initiate two RNA pol II transcription events that copy both DNA strands in opposite directions (bi-directional transcription)

Question 59

What does eRNA transcription do?

Answer 59

eRNA may be contributing to opening up the chromatin of the ACH rather than producing a functional transcript.

Question 60

Which part of the ACH is active?

Answer 60

The whole ACH is a site of active transcription.

Question 61

How does replication occur within chromatin?

Answer 61

Replication of linear eukaryotic chromosomes initiates from multiple replication origins (ORIs) which initiate bi-directional replication forks that eventually meet up to complete the replication process

Question 62

What is sort-seq used for?

Answer 62

Sorting cells according to cell-cycle stage and sequencing their genomes using NGS, replication origins can be mapped because they yield double the number of reads to the background genome in S-phase

Question 63

Where do ORIs occur?

Answer 63

They can occur in euchromatin and heterochromatin and close to or within activated or repressed gene-regulatory regions. ORIs do not have conserved sequences.

Question 64

What are yeast ORIs often called and give an example?

Answer 64

Autonomously Replicating Sequences (ARSs) e.g. the ARS106 ORI is located within the upstream activation sequences responsible for the divergent transcription go the constitutively expressed CYC3 and CDC19. NOTE: yeast ORIs have conserved sequences unlike mammalian ORIs.

Question 65

What is the supposed reason for placing ORIs within gene-regulatory DNA?

Answer 65

To make use of the local chromatin-remodelling activities that are recruited by TFs

Question 66

Where are YEAST ORIs/ARSs found specifically?

Answer 66

In nucleosome-free regions and often close to binding motifs of the Abf1p transcription factor

Question 67

What role does the Fun30 CRA (a SWI/SNF class ATPase) have?

Answer 67

Fun30 CRA is required for normal ORI/ARS and UPE nucleosome position in yeast

Question 68

What are eRNAs ultimately degraded by?

Answer 68

RNases

Question 69

What makes mRNA special?

Answer 69

Most higher eukaryotic pre-mRNA transcripts have introns which the spicing machinery senses and removes and in the process attaches proteins that make the mRNA stable

Question 70

What are the three types of general organisation of human gene regulatory regions?

Answer 70

1) Upstream promoter elements only
2) UPE plus widely separated enhancers/LCRs
3) UPE plus super-enhancer

Question 71

What are super-enhancers?

Answer 71

Extended arrays of TF-binding motifs which act as both LCRs and enhancers and form looped ACH interactions too (rare in human/mammalian genes)

Question 72

What are TADs?

Answer 72

Topologically associated domains are a general association of one particular group of chromosomal loci with each over >1Mbp scale

Question 73

What sets the boundaries for TADs?

Answer 73

Just like ACHs, TAD boundaries are set by insulators that bind CTCF and cohesin

Question 74

Describe the two types of TADs.

Answer 74

1) Transcriptionally active TADs which exhibit gene-active histone PTMs, consist of active genes/active chromatin hubs and replicate early in S-phase = “euchromatin”
2) Transcriptionally inactive TADs which exhibit silent-gene histone PTMs, consist of inactive genes (or are gene-poor) and replicate late during S-phase = “heterochromatin”

Question 75

ACHs are sometimes referred to as…

Answer 75

…sub-TADs

Question 76

What method can be used to reconstruct the physical structure and positioning of chromosomes and sequences within an interphase nucleus?

Answer 76

Contact probabilities from 3C experiments

Question 77

What have 3C and FISH (fluorescent in situ hybridisation) experiments shown?

Answer 77

Individual chromosomes maintain distinct territories within the interphase (transcribing) nucleus

Question 78

Where can we expect to find euchromatin TADs and heterochromatin TADs?

Answer 78

Euchromatin TADs are found in the interior of the nucleus whereas heterochromatin TADs are often at the nuclear periphery (tethered by CTCF). TADs tightly associated with the nuclear lamin proteins at the nuclear periphery are called LADs (Lamin Associated Domains).

Question 79

Describe the hierarchy of structure in the nucleus.

Answer 79

Nucleus –> territories –> TADs –> ACHs –> gene sequence

Question 80

The formation of TADs and sub-TAD/ACH appears to be an intricately organise process however, the latest model suggests that this order within the interphase nucleus is “emergent” rather than following a design or mechanism - explain this

Answer 80

Chromatin domains and locally remodelled regions have distinct TFs, co-activators/repressors, chromatin PTMs and transcripts = “factors”. Current theory is that together these “factors” have common biophysical properties that cause nucleosome chains to condense into distinct chromatin phases which we see as TADs and ACHs.

Question 81

Does TAD organisation change?

Answer 81

Overall TAD organisation is stable between cell types and even between similar mammalian chromosomes however, as gene-regulatory regions are activated or repressed, dynamic changes are observed within TADs reflecting rearrangement of DNA interactions. Individual genes can move between heterochromatin and euchromatin TADs depending on their regulated state e.g. Hox genes move TADs during mouse development, all start off in an active TAD and as they move some become inactive and form a new active TAD

Question 82

How does TAD organisation change?

Answer 82

Genes may re-arrange between TADs, due to gene activation/repression, via phase separation as their chromatin factors change

Question 83

What happens when chromosomes change structure to become condensed during metaphase?

Answer 83

The TAD level of organisation disappears during M-phase chromosome condensation. Virtually all transcription ceases; RNA polymerases, co-activators/repressors and most TFs are evicted from M-phase chromosomes however gene regulation information is not completely erased during M-phase, activated pioneer TFs remain bound at LCRs and previous patterns of histone PTM persist.

Question 84

What is the structure of the condensed M-phase chromosome?

Answer 84

Loops of chromatin containing 100kb DNA are associated with a scaffold structure; the loops are condensed with the chromosome. M-phase chromatin proteins are clearly required to condense the loops but histones are not important.

Question 85

What is the role of Aurora and Haspin kinases?

Answer 85

On entry to mitosis, cell-cycle regulated Aurora and Haspin kinases phosphorylate H3 tails at serine and threonine residues to create an M-phase-specific chromatin PTM pattern. Aurora kinase phosphorylates H3S10 and H3S28 whereas the Haspin kinase phosphorylates H3T3. H3S10P recruits an HDAC that deacetylates H4K16, the loss of this acetyl group promotes inter-nucleosomal interaction and could promote aggregation of nucleosome chains to drive M-phase chromosome condensation.

Question 86

Are nucleosomes required for M-phase chromosome condensation?

Answer 86

Histone-free chromosomes happily condense into M-phase chromosomes when placed into M-phase cells. The latest analysis of M-phase chromosomes is again consistent with a locally disordered chain model suggesting that nucleosomes are relatively passive bystanders during condensation of their DNA loops.

Question 87

Which proteins are condensing the metaphase chromosome?

Answer 87

The scaffold of the M-phase chromosome consists of topoisomerase II alpha and condensins I and II

Question 88

What are topoisomerase?

Answer 88

Topoisomerase’s are enzymes present in both prokaryotes and eukaryotes which manipulate DNA topology to control the presence of “plectonemic” chromosome supercoils

Question 89

How does topoisomerase II alpha relax supercoils?

Answer 89

Topoisomerase II alpha uses ATP hydrolysis to relax supercoils by binding two ds DNA strands and passing one through the other. Topoisomerase II alpha seems to be both catalytic and structural within the M-phase chromosome scaffold: it holds DNA strands together and manipulates supercoiling in the loop itself.

Question 90

What are SMC factors and give some examples?

Answer 90

Structural Maintenance of Chromosomes factors. Condensins I and II and cohesins (encircles DNA molecules to glue ACHs, insulators and sister chromatins/centromeres together) are eukaryotic members of the SMC factors..

Question 91

Condensins are also topoisomerase, how do condensins create positive DNA supercoils in vitro?

Answer 91

Condensins have an ATPase activity.

Question 92

What is the likely mechanism of condensins driving chromosome condensation?

Answer 92

The condensins probably create and then encircle and trap supercoils within M-phase chromosome DNA loops to drive chromosome condensation.

Question 93

What is the current two-step model for M-phase chromosome compaction?

Answer 93

1) Linear compression by formation of loops around scaffold
2) Axial shortening to provide compact structure.

In the early stages of M-phase condensin I is cytoplasmic and only condensin II binds DNA in the nucleus however, at nuclear envelope breakdown both become able to bind chromosomes - the sequential binding of the two condensing probably drives the two-step compaction.

Question 94

What does Ki-67 do to metaphase chromosomes?

Answer 94

Ki-67 binds to the periphery of metaphase chromosomes and appears to act as a capsule/surfactant to keep chromosomes separate from each other. Ki-67 is a huge protein, deletion causes mitotic chromosomes to clump together.

Question 95

The structures of both metaphase and interphase chromosomes are probably not following a template or “design”, they are both emergent properties of the biophysics of chromatin chains - describe the structures and how they seem to form.

Answer 95

The M-phase “sausage” emerges from a battle between the formation/dissolution and trapping of supercoiled loops of chromatin.

Interphase territories, TADs and sub-TADs emerge from phase separations of chromatin protein types.

Question 96

What is the ORC complex and what does it do to promoter chromatin accessibility?

Answer 96

The ORC complex is an ORI-binding factor. ORI-binding factors directly recruit replication-specific chromatin-remodelling HAT complexes (such as HBO1) to promote chromatin accessibility. Remodelling is catalysed by complexes shared between transcription and replication, and those unique to replication.

Question 97

How are the TEC and replicase similar when moving along chromatin?

Answer 97

Both elongating RNA pol II (the TEC) and the elongating replicase are huge chromatin processing machines: remodelling, removing and replacing nucleosomes as it moves through chromatin. Many of the same CRAs and covalent chromatin-remodellers used by elongating RNA pol II are also used by the replicase.

Question 98

As well as replicating chromatin as a substrate what other important role does RNA polymerase have?

Answer 98

It may also play a role in replicating potentially epigenetic information such as that written in histone PTMs.

Question 99

What happens to nucleosomes during DNA replication?

Answer 99

The original (“parental”) histone cores are displaced by a combination of the replicase itself and replicase-recruited chromatin-remodelling ATPases (CRA). CRAs of the ISWI and INO80.com classes (also involved in nucleosome dynamics during gene-regulation transcription) are recruited to the moving replicase by PCNA. The original/ parental histone dimers and tetramers are ultimately transferred to the FACT and Asf1 histone chaperones attached to the MCM helicase. Both FACT (facilitates chromatin transcription complex) and Asf1 appear to be attached to the replicase via interactions with the Mcm2-7 helices. FACT is part of the Mcm/GINS/Cdc45 complex in yeast.

Question 100

When are the Asf1 and FACT chaperones used?

Answer 100

The Asf1 and FACT chaperones are used during gene-regulation, transcription and replication.

Question 101

What happens to the “old” histones and what is the problem that arises?

Answer 101

They are recycled onto the new daughter chromosomes however the cell will only have half the amount of histones needed for replication so new histones are also needed.

Question 102

What are the two ways new histone proteins are synthesised?

Answer 102

1) Replicative histones: large amounts of the canonical histones H2A, H2B, H3 and H4 are synthesised and incorporated during S-phase. Multi-copy genes; regulated burst of transcription and translation; mRNAs have no poly(A) tail.
2) Replication-independent histones: variant histone forms are produced at modest levels, and incorporated into chromatin throughout the cell cycle. Single/low-copy genes; constitutive transcription and translation; mRNAs are polyadenylated.
* New replicative histone dimers/tetramers are assembled on, and bound, by histone chaperones too: NAP-1 (nucleosome assembly protein) binds new H2A:H2B dimers. Asf1 (anti-silencing factor) binds new H3:H4 dimers and, once in the nucleus, hands them onto CAF-1 (chromatin assembly factor) where they appear to associate as H3:H4 tetramers

Question 103

What marks a new histone H4?

Answer 103

“New” histone H4 is marked by diacetylation on K5 and K12 by the HAT1 enzyme.

Question 104

How are new-histone:chaperone complexes transported into the nucleus at S-phase?

Answer 104

Specific karyopherins/importins carry a wide range of cargoes into nuclei.

Question 105

Give some examples of karyopherins involved in new histone transport.

Answer 105

Karyopherins involved in new histone transport include Kap114 (H2A and H2B) and Kap123 (H3 and H4)

Question 106

Where do the old and new histones end up?

Answer 106

Both OLD (parental) and NEW (newly synthesised) histone dimer/tetramers end up in the S-phase nucleus and associated with the replicase

Question 107

What role do the histone chaperones CAF-1 and NAP-1 have in nucleosome assembly?

Answer 107

CAF-1 and NAP-1 catalyse nucleosome assembly onto DNA, they act as a nucleosome assembly complex . You can buy purified CAF1 and NAP-1 to assemble histones onto DNA for in vitro experiments. In vivo they recruit chromatin-remodelling ATPases to help assemble and correctly space nucleosomes. In mammalian cells these require ISWI complexes NURF, ACF/CHRAC and RSF an the CHD factor CHD1. CHD1 is also required for nucleosome re-disposition by the RNA pol II and TEC.

Question 108

Which component of the histones is added first during replication?

Answer 108

(H3:H4)2 tetramers - a mix of old and new ones are randomly segregated between each daughter DNA strand. Deposition of H2A:H2B lags behind and also mixes old and new histones.

Question 109

Semi-conservative DNA replication accurately copies DNA sequence. Does semi-conservative histone replication allow faithful copying of the epigenetic PTM codes?

Answer 109

Yes and no.

Yes: H3/H4 PTMs might be heritable through S-phase by both replication linked and un-linked mechanisms. Old H3:H4 tetramers are immediately recycled behind the replication fork thus tagging them with parental PTMs. Immediately recycled methylated H3/H4 residues in silent genes recruit general heterochromatin repressor complexes such as HP1 and Polycomb factors which allow re-establishment by the normal chromatin-spreading mechanism. PTM adding chromatin remodellers contain a domain which recognises the very modification the complex itself catalyses e.g. PRC2 (polycomb repressive complex) creates and recognises H3K27me3. Although diluted by new histones, enough of the original PTM is recycled to the daughter DNA to allow re-establishment. Although re-establishment of the HP1 binding-directed methylation can take place via HP1 binding-directed methylation, this process is also directly linked to the passage of the replicase. Both the replicase and CAF1 directly bind to and recruit components of the HP1 system to the replication fork : “the CAF-1 memory module”. DNMT1 recognises and converts semi-methylated CpG back to its fully methylated form and is linked to PCNA; CAF-1 acts as chaperone for HPI in addition to H3 an H4 and also picks up MBD1 and KMT1E from the parental DNA.

No: Old and new H2A:H2B dimers are deposited slowly and mix as a pool so their PTM information is lost. Gene-regulatory chromatin structures and many histone PTM states are generated by TFs and co-activators/co-repressors independently of DNA replication. Both HP1 and matured chromatin states can also re-establish themselves eventually using replication-independent mechanisms.

Question 110

What is meant by the term labile chromatin?

Answer 110

Newly replicated chromatin is structurally labile and requires maturation. Newly replicated chromatin retains synthesis-specific PTMs such as H5K5K12ac2 and is highly sensitive to added DNase I indicating a labile structure.

Question 111

What is maturation?

Answer 111

Chromatin maturation leaves stable nucleosomes, as measured by reduced accessibility to DNase I digestion. This process involves nucleosome spacing by ISWI class ATPases. CRAs such as ACF/CHRAC and RSF, which contain ISWI class Atlases, establish regular spacing between newly deposited nucleosomes. The ISWI enzyme seems to contain a molecular ruler as part of its structure. Maturation also involved deacetylation by HDAC1.

Question 112

Removal of which molecules from the replicase allows the recruitment of the chromatin maturation factors?

Answer 112

PCNA molecules.

Question 113

Mutations in which two complexes delay effective gene silencing by heterochromatin after replication?

Answer 113

ISWI and HDAC1. This suggests that these factors contribute to a replication-dependent re-establishment of chromatin state too.

Question 114

Is chromatin TRUELY epigenetic?

Answer 114

The strictest definition posits that information outside of DNA base order is only epigenetic if it is heritable across both mitosis and meiosis i.e. is transgenerational. Most mammalian histone PTMs and gene-regulatory chromatin structures are wiped clean twice during development: during gamete formation and just after fertilisation. Chromatin states may therefore just be relatively stable rather than permanently heritable.