Cell nucleus

Chromatin structure

Gábor Szabó, 2020

Chromatin, nucleus, nuclear membrane/transport:

Essential 3rd ed.:

Pages:16-17, 171-196, 240-47, 272-81, 498-9, 502-505

Essential, 4th ed.:

pages 180-195, 203-207, 230-238, 270-284, 488-497

• + lecture material: most slides of this and of the next

lecture are important!

• Extra material on the web!!!

• Where considered useful, links to Lodish or Alberts

were included

Keywords (nucleus, chromatin,

regulation of gene expression)

Barr body

condensins, cohesins

chromosome territories

transcription factories

replication factories

DNase I hypersensitive sites

DNase I sensitive regions

epigenome

histon-code

DNA methylation

posttranslational modifications of histones

euchromatin

constitutive and facultative heterochromatin

FISH (fluorescent in situ hybridization)

histone “tails”

nucleosomes

nucleosome positioning

kinetochore

nuclear lamina

lamin binding receptor (LBR)

nuclear matrix (nucleoskeleton)

PEV (position effect variegation)

R bands

repetitive sequences

superhelical structure

topoisomerase enzymes

transcription factors

promoter

enhancer

transcription

posttranscriptional processing

splicing

beads-on-a-string

nucleolus

Structure of nucleus and its main functions

Staining with fluorescent dyes

Visualization of main functions by nucleoside analogue incorporation

Evolutionary significance of having a separate nucleus

Homefields

Who flies above, has the sight of a map,

Invisible remains that tired worker’s hat,

He sees fields and wide landscapes,

But not this old woman there, crying for her cat… (M. Radnóti)

(Loewenhook,

1719)

Robert Brown, 1831

info

2-3% of the nuclear volume

is occupied by DNA (or

chromatin)

!

yeast

cell

liver

cell

fibroblast

epithelial

cell

info

Structure of the Nucleus

• Chromatin (strong staining: Giemsa,

hematoxylin-eosin, fluorescent dyes; 3H-

thymidin, BrdU incorporation

• Suborganelles: nucleoli, nuclear bodies

• Double nuclear membrane

• Nuclear matrix

!

Functions of the nucleus

• stores and protects the chromosomes

i.e. the genetic material:

• transports regulatory factors & gene products via nuclear pores

• produces messages that code for proteins (transcription into mRNA)

• DNA replication

• produces ribosomes in the nucleolus

• DNA repair and recombination

Walter Sutton (left) and Theodor Boveri (right)

independently developed the chromosome theory of

inheritance in 1902.

Resting

nuclei

Nuclei

synthesiz-

ing DNA

Cytoplasm

Hoechst, DAPI

Nucleotide (3H-thymidine) incorporation

autoradiography

!

DNA

replication

Hoechst BrdU+anti-BrdU

BrdU ~ thymidine

nucleoside→membrane transit → +3P → BrdU-TP →

→ incorporation during DNA replication

!DNA

replication

Transcription

info

RNA-specific dye

REVISION

Role of nuclear compartmentalization

• Sterical separation of transcription and translation

• Development or subsistence of exon-intron structure of

genes what allows for a free combination of exons, i.e. of

functional building blocks of proteins →engine of the

evolution of the eukaryotic world

• Sterical separation of signaling

steps

REVISION

DNA: amount, single-copy and repetitive, molecular types

DNA content of different cells:

info

The diploid human somatic cell

contains:

• ~ 6.2 pg DNA

• ~ 6x109 bp

• 46 chromosomes

• 4 cm DNA in a chromosome (average)

• ~ 2 m DNA in a cell

• Degree of compactation: ~10,000x

!

Tm, reassociation kinetics,

hybridization, repetitive

sequences

Reassociation kinetics depend

on the repetitive fraction

present in the (fragmented)

DNA sample

DNA

info

Hairpin

etc.

R-loop

Irregular (non-B) DNA structures: physiological and pathological functions

(translocations?)

G4-DNA

info

DNA

Chromatin: categories, hierarchical structure, topological features,

enzymes controlling topology

Chromatin

• DNA, histones,

nonhistone proteins

(regulatory proteins,

transcription,

replication and repair

apparatus)

• euchromatin and

heterochromatin

– constitutive

– facultative

• nucleoplasm

!

Hierarchical levels of chromatin architecture

average size of exons

!

!

The basic unit of chromatin:

nucleosome

• The “core” contains 8 histones wrapped

around by 146 bp DNA (2-2 molecules of

histones H2A, H2B, H3 and H4)

• H1 binds to internucleosomal linker DNA

• ~ 200 bp DNA / (nucleosome+linker)

!

Low ionic

strength,

no

bivalents,

no H1

!

info

Nucleosomal packaging

requires negative

superhelicity

Gene regulatory role?

Possible superhelical states of a

circular plasmid

topoisomerase

info

Superhelicity changes in the course of transcription

Relaxation by topoisomerases

!

Topoisomerasestheir poisons: used as cytotoxic agents

Top I: single-strand cleavage and religation

Camptothecin (CPT): Top I poison

!

Top II: ds

cleavage and

religation

Chromosoma, September 2013

Effect of Top II

deficiency or

inhibition:

anaphase block

!

Chromatin, higher-order organization:

30 nm fiber

100 kb loops (Mbp domains: not discussed)

Chromosome territories and experimental evidence of their existence

Electron spectroscopic analysis: 10 nm fibers

also within heterochromatin.

info

...compact two-start helical fibre structures with stacked alternating nucleosomes

are consistent with RICC-seq fragmentation patterns from H3K9me3-marked

chromatin, while non-compact structures and solenoid structures are consistent

with open chromatin. (Nature 2017, 541: 237–241)

!

Chromatin loops

!

„nuclear halo”

(2 M salt + intercalator)

loops pop out

Ethidium bromide

intercalates between the

basepairs of the DNA

info

5’

Lamin B

Nuclear „halo”

50-150 kb loops, anchored at

S/MAR (scaffold/matrix*-associated)

DNA sequences to the

ribonucleoprotein nuclear matrix

!

*lamina + ??

Compartmentalization

in the nucleus

!

Interphase

R-bandsG-,.Q-bands

EuchromatinHeterochromatin

Relationship between interphase and metaphase

compartmentalization

!

Possible arrangement of chromosomes in the

interphase nucleus

„Chromosome territories”

anti-BrdU

FISH

……..

Interphase chromosome territories

„halo”nucleus

BrdU-pulse

Cell

division

!

Nucleolus, nuclear bodies

Nucleolus: FC, GC, DFC: Fibrillar Center, Granular Component,

Dense Fibrillar Component

info

FC

GC

Nuclear bodies

(suborganells)

• EM: nucleolus

• EM ill. (auto)antibodies:

– speckle, paraspeckle

– coiled body (Cajal)

– histone body

– PML, PML/RAR

– polycomb body

– repair foci

– Transcription and

replication factories

snoRNP

Cajal

IGC

info