MBGE 206 CH 8

nucleosome

The basic structural subunit of chromatin, consisting of about 200 bp of DNA and an octamer of histone proteins

histone tails

Flexible amino- or carboxy-terminal regions of the core histones that extend beyond the surface of the nucleosome.– Histone tails are sites of extensive posttranslational modification.

10 nm fiber

A linear array of nucleosomes generated by unfolding from the natural condition of chromatin

linker histones

A family of histones (such as histone H1) that are not components of the nucleosome core.– Linker histones bind nucleosomes and/or linker DNA and promote 30 nm fiber formation.

30 nm fiber

Coil of nucleosomes. It is the basic level of organization of nucleosomes in chromatin.

nonhistone

Any structural protein found in a chromosome except one of the histones.

Organizational hierarchy

Histones wrap DNA aorund them to form nucleosome beads. Nucleosomes strung together make up 10 nm fiber. INteractions between nucleosomes cause it to fold into this thicker fiber called 30 nm fiber. 30 nm fiber form loop domains that attach to proteins called 300 nm fiber and further coiling of 300 nm fiber forms metaphase chromosomes which are 700 nm thick

MNase

cleaves linker DNA and releases individual nucleosomes from chromatin. More than 95% of the DNA is recovered in nucleosomes or multimers when MNase cleaves DNA in chromatin

length of DNA per nucleosome

The length of DNA per nucleosome varies for individual tissues or species in a range from 154 to 260 bp.

subtypes of nucleosomal DNA

Nucleosomal DNA is divided into the core DNA and linker DNA depending on its susceptibility to MNase. The core DNA is the length of 145–147 bp that is found on the core particles produced by prolonged digestion with MNase. Linker DNA is the region of 7 to 115 bp that is susceptible to early cleavage by nucleases

Structure of nucleosome

A nucleosome contains approximately 200 bp of DNA and two copies of each core histone (H2A, H2B, H3, and H4). DNA is wrapped around the outside surface of the protein octamer. The histone octamer has a structure of an H32-H42 tetramer associated with two H2A-H2B dimers

which histone is associated with linker DNA

H1 is associated with linker DNA and can lie at the point where DNA enters or exits the nucleosome.

Interactions of histones

Each histone is extensively interdigitated with its partner. All core histones have the structural motif of the histone fold. N- and C-terminal histone tails extend out of the nucleosome.

Modificaitons seen on histones

Histones are modified by methylation, acetylation, phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, and other modifications.

Histone Code Hypothesis

Combinations of specific histone modifications define the function of local regions of chromatin

Code-reader complex

Complex made up of scaffold protein + subunit protein modules to recognise specific histone modifications on nucleosome. Binds and recruits other components.

How are histone modifications spread around in genome?

Regulatory protein + writer protein complex come and catalyze the modification. Then code-reader protein comes and recognizes the modification and extends its other end to the other nucleosome, where writer protein binds and modifies. (Reader - writer complex). ATP-dependent chromatinn remodeling complex can also interact with reader-writer complex.

How are neighbouring chromatin domains separated?

By barrier proteins insulating interactions

How are nucleosomes modified?

Covalently

Histone variants

can be closely related to or highly divergent from canonical histones. Different variants serve different functions in the cell. All core histones except H4 are members of families of related variants.

Structures 30 nm fibers can be organized into

either a one-start solenoid or a two start helix

factors that promote secondary structures such as 30 nm fiber

Histone H1, histone tails, and increased ionic strength all promote the formation of secondary structures

Higher order 3D structures

Secondary chromatin fibers are folded into higher order, three-dimensional structures that comprise interphase or mitotic chromosomes

Which unit of the structure is conserevd during replication?

Histone octamers are not conserved during replication, but H2A-H2B dimers and H32-H42 tetramers are.

Sıralama in formation of nucleosome

During nucleosome assembly in vivo, H3-H4 tetramers form and bind DNA first, then two H2A-H2B dimers are added to form the complete nucleosome.

Proteins in nucleosome assembly

Accessory proteins are required to assist the assembly of nucleosomes.

CAF-1 and ASF1 are histone assembly proteins that are linked to the replication machinery.

HIRA, and the histone H3.3 variant are used for replication-independent assembly.

Measures taken through nucleosomes for replication

Replication fork passage displaces histone octamers from DNA.

how are nucleosomes placed?

Nucleosomes can form at specific positions as the result of either the local structure of DNA or proteins that interact with specific sequences

INDIRECT END LABELING

A technique for examining the organization of DNA by making a cut at a specific site and identifying all fragments containing the sequence adjacent to one side of the cut.– It reveals the distance from the cut to the next break(s) in DNA.

Cut from next to hypersensitive site by DNase I, extract DNA sequence and cleave from restriction site, gel electrophoresis to check for fragment size

Relation of transcription and nucleosome organization

Most transcribed genes retain a nucleosomal structure, though the organization of the chromatin changes during transcription.

Some heavily transcribed genes appear to be exceptional cases that are devoid of nucleosomes.

RNA polymerase displaces histone octamers during transcription in vitro, but octamers reassociate with DNA as soon as the polymerase has passed

proteins needed for nucleosome disassembly / reassembly

Additional factors are required both for RNA polymerase to displace octamers during transcription and for the histones to reassemble into nucleosomes after transcription

effect of trasncription on nucleosome organization

nucleosome reorganization (disassembly and reassembly)

hypersensitive sites

found at the promoters of expressed genes as well as at other important sites such as origins of replication and centromeres. Any hypersensitive site is an accessible site. Hypersensitive sites are generated by the binding of factors that exclude histone octamers

how are domains with transcribed genes identified?

A domain containing a transcribed gene is defined by increased sensitivity to degradation by DNase I.

TAD dynamicity

Mammalian chromosomes are organized as strings of topologically associated domains (TADs) that average about 1 megabase (Mb) in size.

TADs or TAD-like structures have been found in most eukaryotes.

Loci within a TAD interact frequently with each other, but less frequently with loci in an adjacent TAD.

TAD organization is fairly stable between cells, but interactions within TADs are highly dynamic

How are TADs separated

Boundary regions between TADs contain insulator elements that are able to block passage of any activating or inactivating effects from enhancers, silencers, and other control elements

Insulators can provide barriers against the spread of heterochromatin.

Insulators are specialized chromatin structures that typically contain hypersensitive sites.