Week 8: Chromatin + Nuclear Organization

DNA organization

• 2m of DNA compacted to fit into nucleus

• 1st order = 10nm chromatin fibre

  • in vivo → all 10nm

  • packaged w/o 30nm fibres into mitotic chromosomes

  • regulated w/o changes b/w 10-30nm fibres

• 30nm fibres + higher-order fibres = artifact of in vitro purification

• DNA wraps around core histone octamer protein complex → forms nucleosome

nucleosome

fundamental structural unit of chromatin

• DNA-histone complex

  • DNA wraps ~1.6 turns around histone complex

  • histone proteins = H2A/H2B/H3/H4 → 2x of each form octamer

    • mostly a-helices

    • tails sticking out can be modified → recognized by other proteins

histone

(+) charged protein core of nucleosome + wrapped by DNA

• make tight but not sequence specific contact b/w DNA + (+) charged proteins

• has acidic (-) patch → no interactions w/ DNA

  • drives orientation

• tails are flexible unstructured domains

  • stick out from nucleosome

  • can be post-translationally modified to alter gene expression

• basic (+ charged) properties help neutralize (-) charge of DNA phosphate backbone

chromatin properties

• (-) charge of phosphate-sugar backbone stabilized by (+) charges of histone proteins

• reduce steric repulsion + enables bending + flexible polymer properties

naked DNA properties

• periodic structure w/ regularly spaced (-) charge from phosphate backbone

• requires metal ions + small amino-rich molecules to neutralize charge + enable flexibility

post-translational modifications

contributes to gene regulation

1. DNA methylation = changes TF recruitment → changes gene expression + DNA metabolism

   1. A + C methylated more than G + T

      1. eukaryote → 5% of cytidine residues methylated → common at CpG sites

      2. prokaryotes → adenine commonly methylated

   2. DNA methylases use S-adenosylmethionine (SAM) as methyl donor

2. modification to histone tails = changes nucleosome packaging = changes gene expression

   1. nucleosomes moved out of way so polymerase can go to site

      1. DNA needs to be bent at promoter site

      2. open = active chromatin

      3. closed = inactive chromatin

   2. provides platform for recruiting protein complexes → activate/silence particular region of genome

   3. in combo w/ DNA methylation → mark specific genes for activation/repression

chromatin

complex of DNA and proteins found in eukaryotic cells

• package long DNA molecules into more compact, denser structures

• made up of string of nucleosomes attached by linker DNA

• 10nm = in vivo

• 30nm+ = in vitro artifacts

• needs to be “open” for proteins to reach genetic material

karyotyping

technique that visually assesses # and structure of chromosomes in cell, identifying abnormalities related to genetic disorders

• length + staining density of genomic regions assessed w/ light microscopy

1. arrest cell in cell cycle

2. isolate DNA from cells

3. add methanol + other things

4. take mixture + drop onto slide

5. mitotic chromosomes spread out into little splats

6. different stains used to look at them

FISH (fluorescence in situ hybridization)

method to visualize/detect specific sequences of DNA → where segments of genome are

1. fluorescent probes made from specific DNA + cut into small fragments

2. cells fixed + chromatin chemically denatured → heated to melt DNA

3. fluorescent probes annealed + non-specific probes washed away

4. detection via fluorescence microscope

interphase chromosomes

chromatin from individual chromosomes form distinct domains

• fibres within territories are 10-nm

  • strings of nucleosomes attached by linker sequences

• epigenetic modifications may change local chromatin environment

  • change space b/w nucleosomes

  • provide platform for protein/protein-RNA complexes to bind + regulate gene expression

chromatin in situ

• interphase

  • chromatin organized into compact discrete structures → chromosome territory

    • fibers in territories = 10-nm chromatin

      • strings of nucleosomes attached by linker sequences

• epigenetic modifications may change local chromatin environment

  • change nucleosome spacing

  • provide platform for proteins/protein-RNA complexes

cell cycle chromatin organization

interphase (G1/S/G2) = cell grows + DNA replicates

• genes packaged in looser, more accessible structure

  • non-transcribed regions more condensed, less accessible

• DNA + histone modifications

• chromatin form chromosome territories

mitosis = cell division

• chromatin condenses → chromosomes

  • organized for efficient segregation to separate

nuclear bodies

• range in size + function → large nucleolus to small polycomb bodies

• non-membrane bound sub-nuclear compartments w/ specific function

  • eg. nucleolus = ribosome biogenesis

• spatial + temporal regulation of nuclear processes

• sequestration of molecules improves reaction efficiency

• active genomic regions → clustered around transcription factories w/ active RNA polymerase

  • can be visualized w/ fluorescent microscopy

protein phase separation

process where proteins dynamically aggregate to form membrane-less compartments, influencing various cellular functions and processes

• some proteins + RNA under certain conditions self-solubilize → form phase-separated compartment

  • eg. concentration, post-translational modification

1. interlocking proteins via multivalent domains

   1. proteins post-translationally modified → drives association or dissociation

   2. [sufficient] → spontaneous formation of membrane-less compartments

   3. proteins can interlock

2. pi-stacking via disordered/low-complexity regions

   1. phenylalanine rings + glycine build up force → F-G repeats

      1. need to be on flexible part of protein to stack

fluorescence microscopy

way to visualize/detect proteins

1. antibodies raised to detect protein of interest or protein of interest tagged w/ fluorescent protein, eg. GFP

2. cells fixed + permeabilized → antibodies can fit into cellular compartments

3. fluorescent-tagged secondary antibody often used

4. DNA counterstains, ie. DAPI + Hoescht, define nucleus

   1. give sense of regions of more or less DNA → compaction/heterochromatin

progeria

rare genetic disorder resulting in rapid againg

• caused by point mutation in lamin A

  • de novo (meiotic polymerase replication) error causes C → T mutation in exon 11

  • generates cryptic splice site

  • 1 allele expresses truncated form of lamin A

    • 50 AA shorter than WT

lamin A RNA

• affected individual has both full-length + truncated version of lamin A mRNA

  • WT = 639 bp

  • mutant = 489 bp

lamin A protein

• lamin C reacts w/ antibody for lamin A

• affected individual has small amount of alternatively spliced version

  • WT (2/3/6/7)

  • mutant (1/4/5) = del 50 prelamin A variant band smaller than WT

progeria cells

control (A-D)

• nucleus = smooth, oval shape

• lamin localizes to periphery, some in nucleoplasm

mutant (E-H)

• nucleus = reticulated + irregular

• lamin localizes throughout nucleoplasm

green LaminA; blue DAPI; red mitochondria

progeria cell treatment

• progeria Lamin slow to recover compared to WT

  • ⬇ mobility of Lamin A protein

• WT dynamics restores by ⬆ oligo (nucleic acid = small RNA molecule) to progeria cells

  • ⬇ amount of alternatively spliced Lamin A mRNA

  • oligo targets cryptic exon 11/12 boundary → sends RNA for degradation

FRAP (fluorescence recovery after photobleaching)

measures recovery of fluorescence after being bleached

• determines mobility + dynamics of tagged proteins in live cells

  • often tagged w/ GFP

• recovery time ⬆ = dynamic ⬆