ORGANIZATION AND PACKAGING OF CHROMOSOMAL DNA

Review of cell cycle: majority of cell cycle is interphase Figure 17-3 Molecular Biology of the Cell (© Garland Science 2008)

For most of the cell cycle, interphase, the chromatin is in a relatively extended form, whereas it is most highly condensed in mitosis Figure 4-17 AND 4-18 Molecular Biology of the Cell (© Garland Science 2015)

Three types of DNA sequences are required to produce a eukaryotic chromosome that can be replicated and then segregated at mitosis Figure 4-19 Molecular Biology of the Cell (© Garland Science 2008)

Sister chromatids attached Mitotic chromosome at metaphase: each sister chromatid contains one of two identical sister DNA molecules generated by DNA replication Centric/centromeric heterochromatin Condensed Replicated Sister chromatids attached

Multiple techniques have been developed for visualizing chromosomes— Determine chromosomal abnormalities associated with particular diseases Determine that chromosomes localize to subnuclear domains, even in interphase cells

Giemsa-stained early mitotic chromosomes gives a characteristic banding pattern The arrangement of the full chromosome set in numerical order is called a karyotype Figure 4-11 Molecular Biology of the Cell (© Garland Science 2008)

(highly compacted chromosomes from a cell undergoing mitosis) Chromosome painting or “spectral karyotyping” stains each chromosome a different color (highly compacted chromosomes from a cell undergoing mitosis)

Spectral karyotyping: less compacted chromosomes from a cell in interphase

THE GLOBAL ORGANIZATION OF DNA

Chromatin consists of DNA bound to both histone and non-histone proteins. The mass of histone protein is about equal to total mass of non-histone protein. In total, a chromosome is about 1/3 DNA and 2/3 protein by mass.

Arrangement of genes in the genome of humans compared to yeast S Arrangement of genes in the genome of humans compared to yeast S. cerevisiae The human genome is less densely packed with much more interspersed repetitive DNA sequences between genes that code for proteins or functional RNAs.

Organization of genes on a human chromosome-- Only a small portion of the DNA encodes a protein Figure 4-15 Molecular Biology of the Cell (© Garland Science 2008)

Small amount of human DNA codes for proteins or functional RNA Small amount of human DNA codes for proteins or functional RNA. Over half consists of repetitive sequences; the remainder is intron or regulatory sequences Figure 4-17 Molecular Biology of the Cell (© Garland Science 2008)

The complexity of eukaryotic genomes results from the abundance of different types of coding and non coding DNA sequences. About half of all genomic DNA is repetitive sequences. Genes which code for proteins or functional RNA make up a small percentage of human DNA Genes are comprised of exons, introns (usually) and regulatory regions

Histones and Chromatin Remodeling THE FORMATION OF NUCLEOSOMES AND HIGHER ORDERS OF CHROMOSOME STRUCTURE: Histones and Chromatin Remodeling

DNA is packaged into a highly organized structure, even in interphase Interphase DNA Figure 4-72 Molecular Biology of the Cell (© Garland Science 2008)

Nucleosomes (comprised of DNA and histone proteins) are the basic subunit of DNA organization

DNA is packaged into nucleosomes; two each of core histones H2A, H2B, H3 and H4 plus 147bp of DNA Figure 4-23 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)

The nucleosome core particle from X-ray crystal structure Figure 4-24 Molecular Biology of the Cell (© Garland Science 2008)

Histone proteins have a histone fold and tails subject to covalent modification that can affect chromatin structure and function. Figure 4-25 Molecular Biology of the Cell (© Garland Science 2008)

H3 and H4 form dimers; H3/H4 dimers form a tetramer H2A and H2B form dimers Figure 4-26 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

H3/H4 tetramer is at the center of the nucleosome core particle H2A/H2B dimers then bind to the H3/H4 tetramer Figure 4-26 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)

Nucleosome structure is very stable, but it is not static Figure 4-24 Molecular Biology of the Cell (© Garland Science 2008)

Nucleosomes are dynamic, and are often changed by ATP-dependent chromatin remodeling complexes: one type mediates nucleosome sliding Figure 4-29 Molecular Biology of the Cell (© Garland Science 2008)

Exchange or removal of histone proteins by a different type of ATP-dependent chromatin remodeling complexes Figure 4-30 Molecular Biology of the Cell (© Garland Science 2008)

Interphase nuclei: predominantly in 30nm fiber form of chromatin Interphase nuclei: predominantly in 30nm fiber form of chromatin. This form seems to look “unorganized” compared to mitotic chromosome chromatin. 30 nm fiber “beads on a string” Figure 4-22 Molecular Biology of the Cell (© Garland Science 2008)

Nucleosomes are usually packed together in a compact chromatin fiber (30nm fiber). Figure 4-31 Molecular Biology of the Cell (© Garland Science 2008)

30 nm fiber compaction is in part mediated by inter-nucleosome histone tail interactions. Figure 4-33b Molecular Biology of the Cell (© Garland Science 2008)

30 nm fiber compaction is in part mediated by the linker histone H1.

How does the interphase chromatin keep from getting tangled? A fractal globule model for interphase chromatin knot-free conformation that permits dense packing, and retains ability to easily fold and unfold any genomic locus 5x106

Less compacted DNA is more “active” Interphase DNA More compacted Figure 4-72 Molecular Biology of the Cell (© Garland Science 2008)

Specific genes on the chromosomes can move in response to different stimuli: highly active genes tend to localize to the interior of the nucleus, whereas genes in silenced heterochromatin tend to localize to the nuclear periphery.

Chromosome structure - nucleosomes basic structural unit - histone-DNA interactions  packaging - higher order chromosome structure Chromosome structure is dynamic; -decondensed DNA is more metabolically active than highly condensed DNA. - nucleosome positioning dynamic - ATPase remodeling complexes

THE REGULATION OF CHROMATIN STRUCTURE/FUNCTION THROUGH Short break…then: THE REGULATION OF CHROMATIN STRUCTURE/FUNCTION THROUGH EPIGENETICS

Difference between genetic and epigenetic inheritance. Genetic involves changes in DNA sequence Epigenetics does not change DNA sequence, but changes other properties of the chromatin. Figure 4-35 Molecular Biology of the Cell (© Garland Science 2008)

Heterochromatin highly compacted and is resistant to gene expression Heterochromatin highly compacted and is resistant to gene expression. Genes relocated to heterochromatin are silenced Figure 4-37 Molecular Biology of the Cell (© Garland Science 2008)

During development, heterochromatin can spread; the resulting state of heterochromatin/euchromatin is inherited stably by daughter cells. Heterochromatin is prevented from spreading into adjacent euchromatin by special boundary DNA sequences.

Models for barrier proteins blocking the spread of heterochromatin to adjacent euchromatin regions

Different covalent modifications or combinations of modifications of amino acids in histone protein “tails” affect chromosome structure in different ways, with predictable consequences on various processes Figure 4-39a Molecular Biology of the Cell (© Garland Science 2008)

Many different histone tail post-translational modifications, such as acetylation, methylation, phosphorylation and ubiquitylation have been observed.

decrease positive charge; less tight interaction with DNA Different covalent modifications or combinations of modifications of amino acids in histone protein “tails” affect chromosome structure in different ways, with predictable consequences Lysine acetylation: decrease positive charge; less tight interaction with DNA Lysine methylation: not affect charge; different protein complexes recruited Figure 4-38 Molecular Biology of the Cell (© Garland Science 2008)

“Histone code”—combinations of modifications have different meanings Figure 4-44b Molecular Biology of the Cell (© Garland Science 2008)

The combination of histone tail modifications attracts specific “reader” and “writer” proteins, which execute appropriate functions

How recruitment of a reader-writer complex can spread chromatin changes along a chromosome

Histone variants also affect chromosome structure Figure 4-41 Molecular Biology of the Cell (© Garland Science 2008)

Centromere-specific histone variant of H3 contributes to centromere formation Figure 4-48 Molecular Biology of the Cell (© Garland Science 2008)

Different covalent modifications or combinations of modifications of amino acids in histone protein “tails” affect chromosome structure in different ways, with predictable consequences on DNA metabolic processes. - histone code “writers” and “readers” Variant histones contribute to specialized functions Epigenetic modifications tend to be maintained after DNA replication

Heterochromatin either contains no genes (centromeres and telomeres) or is resistant to gene expression. Multiple types of heterochromatin Heterochromatin is both dynamic and self-sustaining once formed.

Clinical use of chromatin/histone modifying agents

Assessment of Histone Acetylation Immunohistochemical staining of formalin-fixed, paraffin-embedded sections using antibodies directed against acetylated histone H2B lysine 5, or acetylated histone H4 lysine 8. Conclusion: Vorinostat monotherapy is well tolerated in patients with recurrent GBM and has modest single-agent activity. Histone acetylation analysis indicate that target pathways are affected with this dose and schedule. Additional testing of vorinostat in combination regimens is warranted.

Some novel drugs in preclinical or clinical development targeting components of epigenetic machinery Plass et al, Nature Rev Genet 14:765, 2013