(1) Chromosome And Chromatin

Chromosome, Chromatin and Telomere Jiemin Wong, Ph.D. Associate Professor Department of Molecular and Cellular Biology Baylor College of Medicine Houston TX 77030 [email protected] 713-798-6291 (phone) 713-790-1275 (fax)

I. Chromosome and Chromatin II. Modulation of Chromatin Structure III. Telomere and its Implication in Aging and Cancer

I. Chromosome and Chromatin Eukaryotic DNA is packaged into a set of chromosomes Why compaction of DNA into chromosomes is essential?

Genomes and Gene Number

Gene  number

6000

19,000

13,500

30,000

30,000

Simple Calculation Human beings have roughly 3 billion base pairs of DNA in 23 pairs of chromosomes Distance between bases is 3.4 Angstrom For human this would be 3.4x3x109 Angstroms. or 1.02 meters per haploid genome, 2.04size meters pernucleus cell is roughly 10 micrometers The of the in diameter The average size of condensed mitotic human chromosomes is ~ 1 micrometer (>10,000 fold)

If there is no compaction, nucleus would be too small To hold all DNA!!!

Function

• Storage of genetic information • Precise segregation of replicated DNA into two daughter cells • Platform for transcription, replication, recombination and DNA repair

Problem How to retrieve genetic information from DNA packaged into chromosomes?

How the long linear DNA molecules are packaged into compact chromosomes?

Historic View: Chromosomes and Chromatin 1879 : Walter Flemming discovered chromosomes, observing threadlike structures in the nuclei of salamander cells during cell division

Early 1900s, cytologists Walter Sutton and Theodor Boveri, independently published papers linking chromosomes to the Mendelian principles of segregation and independent assortment. Their work inspired the chromosomal theory of inheritance This theory states that hereditary information is on genes and that genes are located on chromosomes

Human cells contain 23 pairs of Chromosomes. For each pair of chromosomes, one is maternal and one is paternal – homologous chromosomes Sex chromosomes are nonhomologous chromosomes, X from mom, Y from dad.

Chromosomes are typically stained by dyes that distinguish between areas rich in A-T nucleotide pairs and areas rich in C-G pairs. This results in a pattern of banding that is unique to each chromosome. Cytogeneticists use these to detect major chromosomal centromere abnormalities.

large rRNA

Compaction of chromatin is cell-stage dependent A. Interphase chromatin

B. a mitotic chromosome, which is duplicated already

Question: How this compaction is achieved?

Compaction of chromatin

Nucleosome is the repeating unit of chromatin  1974

A) 30 nm fibers B) beads on a string­nucleosome From interphase nucleus

Composition of Chromatin DNA Histones (H2A, H2B, H3, H4 and linker histones)

Stable association

Non­histone chromosomal proteins In general not as stable as DNA-histone interactions

Nomenclature Nucleosome = a nucleosome core particle + linker DNA+ a linker histone DNA length: 180-200 bp Nucleosome core particle = histone octamer + 146 bp DNA

Nucleosomes can be  isolated by digesting with nucleases that cut between the nucleosomes in a region called the linker

Nucleosome­octamer 2 each H2A, H2B, H3, H4 142 hydrogen bonds between DNA and nucleosome, mostly between phosphodiester bonds and amino acid backbone of histones

Histones- highly basic (+) proteins Protein

H1

Molecular weight 21

Major Amino acid ++ Lys

H2a

13.8

Lys

H2b

13.8

Lys

H3

15.4

Arg/Lys

H4

11.4

Arg/Lys

Histones – folding and coiling chromosomes – 45% of the total mass – 60 million molecules of each type per cell

Non-histone proteins (NHPs, acidic proteins, nonhistone chromosomal proteins, NHC proteins) – help regulate DNA transcription and replication – at least 30 types

Histone fold­ 3 alpha helices  and 2 folds

N terminal tails are subject to covalent modification­important for transcription

Histone self­assembly

The position of the core histone in the nucleosomes Nucleosome core particle

2.8 A crystal structure of the Mono-nucleosome

From Luger et al Nature 389: 251 - 260 (1997);

Histone tail interactions with DNA

From Luger et al Nature 389: 251 - 260 (1997);

Is DNA in the nucleosome different from DNA in solution? 1. DNA (146 bp) is wrapped in 1.75 left-handed superhelical turns 2. One side of DNA is in contact with histone octamer 3. DNA helical turns in a nucleosome have an average number of base pairs per helical turn of 10.2 vs 10.5 of DNA in solution

Nucleosome Positioning 1. Nucleosome positioning is not random and is defined by translational positioning and rotational positioning. 5S rRNA gene, MMTV LTR and Xenopus TRβA gene promoter 2. The local influences of DNA rigidity and curvature will affect the precise positioning of nucleosomes. Histone octamer prefers binding to AT rich sequence 3. In most cases, nuclosome at given piece of DNA can adopt multiple different positions 4. Nucleosome positioning affects access of transcription factors and other proteins to DNA. The position of nucleosome can allow or disallow the binding of a transcription factor depending whether its binding site is incorporated into the nucleosome or exposed in the linker region

linker

Core particle

Translational positioning

Rotational Positioning

How to determine translational positioning experimentally?

Translational Positioning

180-200bp

Partial Micrococcal Nuclease Digestion

146 bp

Determining sequence

How to determine rotational positioning experimentally?

DNaseI partial digestion Hydroxyl radical cleavage In vitro reconstituted nucleosome End-labeled DNA + Histone octamer

Preferential Cleavage Sites

The effect of Nucleosome Positioning on Transcription p62

SIF

SRF

AP1

CREB/ATF

CTF/NF1­like

DRBP

­30

+1

TATA(a/t)A(a/t)

YYAN(t/a)YY

TATA box

INR

Upstream regulatory elements

Core elements

Highly varied Gene-specific Bound by regulatory proteins

Present in almost all promoters Recognized by basal apparatus

Structure of RNA Polymerase II Promoter

The assembly of DNA into nucleosome has differential effect on binding of transcription factors Sensitive to nucleosome structure: NF1, HSF, TFIIIA, TBP and etc. Not sensitive to nucleosome structure: Gal4, GAGA, thyroid hormone receptor and etc This effect is dependent on nucleosome positioning!

How “the beads on a string” chromatin is folded further into 30 nm chromatin fiber?

Linker histones in higher order chromatin compaction

Histone H1 monomers link nucleosomes

Nucleosomes are further packed into a 30 nm fiber the zigzag model

The 30 nm nucleoprotein fiber Solenoid Model - six to eight nucleosomes per turn

Most interphase chromatin is condensed into 30nm coils. Histone H1 helps this compaction but is not essential!

Further Compaction?

Chromatin in the interphase nucleus is next believed to organized into discrete domains defined by sites of attachment to the nuclear matrix.

Interphase

M phase

Chromosome scaffold

topoisomerase

Histone depleted metaphase chromosomes

Scaffold Attachment Regions (SARs) • Regions of the chromosomes with sequences specific for topoisomerase, HMG protein, and histone H1 binding • Found only in untranscribed regions of the eukaryotic chromosomes • Spaced along the chromosomes, with the intervening regions containing one or more genes? • Highly AT rich (65%) and may be several hundred bp long

The state of condensation of chromosomes varies according to the cell growth cycle. The mitotic chromosomes are highly condensed, in contrast to that of the interphase chromosome.

What structures are necessary to a functional eukaryotic chromosome? • Centromeres • Telomeres • Origins of replication

Three types of specialized sequences found in all eucaryotic chromosomes ensure that chromosomes replicate efficiently.

many, to ensure speed

kinetochore = protein complex that binds the spindle and the centromere

The condensed state is important, allowing the duplicated chromosomes to be separated

Origins of Replication

Centromere

The Functions of Centromeres The centromere is a highly differentiated structure of the chromosome that fulfils a multitude of essential mitotic and meiotic functions •Required for chromosome stability •Sister chromatid pairing •Mitotic and meiotic spindle attachment •Chromosome movement •Cell cycle checkpoint control In most cells, centromeres of mitotic chromosomes that have not yet attained a stable bipolar orientation on the spindle would send a signal to delay the onset of metaphase/anaphase transition

How do Centromeres Work? site of kinetochore formation allowing attachment of the sister chromatids to microtubules emanating from each pole of the spindle kinetochore = large protein complex mediating spindle attachment to chromosomes

The Structure of Centromeres • • • •

One centromere for each chromosome Structurally complex Kinetochore - spindle fiber attachment required for the stability of chromosomes during mitosis • DNA at yeast centromeres is relatively simple • human centromeres is a family of highly repeated, tandemly arrayed ‘satellite’ DNA which measure 300-5,000 kb in length Repeat sequences • Specific associated proteins

Centromere DNA of higher eukaryotes •

The universal presence of a great abundance of tandemly repeated DNA • The size of centromere DNA varies from several hundreds of kilobases to tens of megabases on each chromosome • Lack of sequence conservation!

B. Telomeres

Telomeres: •allow complete replication of the ends of chromosomes •protect them from erosion and fusion with other DNA fragments.

Cell prepares for mitosis makes essential proteins

• Duplicates DNA • Identical sister chromatids • joined at centromere

Mitosis: division of 1 complete set of chromosomes to each progeny nuclei

• prepare for DNA replication • gets large enough to divide • most of time spent in this phase

Interphase Nucleus: euchromatin vs heterochromatin

Interphase chromatin has more- or less-condensed states, depending on how “active” it is, I.e. are the genes being transcribed? A. Euchromatin. – open, dispersed, potentially active – located at the interior of the nucleus – Chromosomes have predictable and unique locations in the nucleus. B. Heterochromatin. during interphase about 10% of the chromatin remains in a compact state similar to the mitotic chromosome. – located at the periphery of the nucleus, looks darker – this DNA is not transcribed or translated 66

– there are two types of heterochromatin: 1. Constitutive heterochromatin, condensed at all times – includes AT-rich satellite DNA at the centromere – the telomeres, or ends of the chromosomes (especially in plants) 2. facultative heterochromatin transient condensation, contains potentially active genes – e.g. less active chromosome: chromosome 18 – e.g. one X chromosome (from mother or father) is “turned off” early in development » this becomes a “Barr body” » in subsequent daughter cells, the same chromosome stays condensed » eventually the Barr body does get reactivated when new germ cells are made – facultative heterochromatin becomes more abundant in cells as the organism matures from embryo to adult, as cells specialize fewer genes are active 22.228 lecutre 7

67

Lampbrush and polytene chromosomes Lampbrush chromosomes: found in the oocytes of many animals. They contain very transcriptionally active DNA, where loops of DNA emerging from an apparently continuous chromosomal axis are coated with RNA polymerase. Each RNA polymerase is attached to nascent RNA and associated proeins generating a visible ‘brush-like’ appearance.

The transcription of the RNA precursor of the 28S, 18S, and 5.8S ribosomal RNAs. These units are tandemly linked together, some 450 per haploid genome.

Polytene Chromosomes of Drosophila

Polytene chromosomes are “giant” chromosomes that have undergone many rounds of DNA duplication without cell division.

Their large size makes them easily visible under the compound light microscope, and makes them amenable for various genetic studies.