Chromosomes and chromatine

structural arrangement of genetic information

Chromatin (Walther Flemming 1882)

= DNA + associated proteins

euchromatin x heterochromatin

heterochromatin facultative x constitutive

Chromatin(DNA + associated proteins)

Genetic information = DNA sequence (change = mutation)

- protein-coding, regulatory, RNA-coding-

Epigenetic information (less stable, depends on location)

- transcriptional activity, access of interacting proteins

transcriptionally active x transcriptionally inactive decondensed, accessible x compact, unaccessible

• DNA methylation• histone posttranslational modifications mutually interconnected! • histone types

Epigenetic modifications of chromatin- epigenetic information can be mitotically and meiotically herritable (e.g. some changes in gene activity)- no change in primary DNA sequence - modifications of chromatin components:

euchromatin heterochromatin

Varying composition:- histone variants (isoforms): CenH3, H3.3, H2A.Z- posttranslational modifications of histone proteins

Nucleosomeoctamer of histones (small alcaline proteins): 2 x H2A, 2 x H2B, (2 x H3, 2 x H4) + 147 bp DNA

Structure of 30nm fibreSolenoid or ZigZag?

- still unclear

solenoid

Li and Reinberg 2011

• LBARs (loop basement attachment regions) - organize chromosomes to huge loops (distances 20kb až 100kb)

• MARs (matrix attachment regions) alt. SARs (scaffold attachment reg.)

– harboring regions surrounding coding sequences to nuclear protein matrix

– AT rich, colocalize with „insulators“(sequences that prevent spreading of heterochromatin)

– distances between 3 - 100kb

Higher structural order of chromatine- hypothetically loops with actively transcribed genes- insufficiently understood

example of hypothetical arrangement

Interconnections betweeen nucleosomes

- linker sequence between: length = multiple of 10 bp (20 to

90 bp)

- average (most frequent) length - differences among species, tissues, …

(20 bp yeast, 30 bp Arabidopsis, 40 bp mammels)

- internucleosomal fragmentation yields: 167 – 237 bp ( frequent length of repeats)

Interconnections betweeen nucleosomes

Direct interactions - N-ends of H4 interact with H2A.H2B bodies in parallel fiber- presence of H2A.Z variant probably prevents parallel

interaction

Linker histone H1 - alcaline both ends (amino and carboxy) interaction with both histones and DNA- stabilization of higher structures (30nm), phosphorylated during cell cycle- length of linker sequence:

longer - require H1, more compact – heterochromatin

shorter – H1 less important, more decondensed, active chromatin

Nucleosome position

• arrangement on DNA is not random (but is changable)

- DNA sequence- DNA methylation- histone modification/types- DNA transcription

• regulation / modulation of transcription- „unstable nucleosom region“ (earlier „nucleosom-free region“) in front of transcription start site (mainly constitutively expressed genes) –

- unstable nucleosomes with H3.3 and H2A.Z histones

- surrounded with stably situated nucleosomes with H2A.Z- nukleosomes help to define exons (central location even without transcription!)

Histone code

- covalent posttranslatinal modifications (PTM)- modifications mainly on N-ends (out of core)- high complexity- „epigenetic instruction“ to manage with DNA

Some histone PTMs are mutually interconnected and have multiple functions

e.g. H2A phosphorylation – injured DNA labelling,but also role in regulation of transcription and spiralizationand in PCD

Rossetto et al. 2012

Histone code

Phosphorylation – predominantly short-time transient label, various functions

Acethylation – predominantly „executive modification“ for weakening interaction with DNA

- K-Ac – specific interaction of bromodomain proteins

Methylation – signal role ( stabile), both repressive and activating (~

depends on position)

- K-Me – specific interaction with chromodomain and TUDOR-

like domain proteins

- key role in regulation of DNA methylation

and chromatine activity

- H3K9me2, H3K4me3, H3K27me3

Reproduction of nucleosomes after replication- histone tetramers (H3/H4) and dimers (H2A.H2B) not divided between sister strands! - one strand – „parental histons“ (Asf1) de novo deposition (CAF1, Asf1) - H2A.H2B incorporated even later (post replication)

Chromosomes

NOR: 18S- 5,8S- 26S rDNA

Caryotype – number, types and sizes of chromosomes

Flow caryotype(FISH labelling)

Doležel et al. 1999

Classical caryotype (metaphase)

Chromosome number and sizes Number: 2 - 600

Size: 2,4 Mb Genlisea

30 Mb Arabidopsis

800 Mb Triticum

What are the consequences?

- different linkage groups (various gene recombination)

- limited hybrid fertility, …

Chromosome number differs between species

• Extreme chromosome numbers – Haplopappus gracilis: 2n = 4– Sedum suaveolens: 2n = cca 640

• Luzula sp.: – 2n = 6 až 66– holocentric chromosomes– Chromosom size differes up to 60x

(Cullis, Plant genomics and proteomics, 2004)

L. pilosa

L. elegans

B chromosomes in plants

- non-pair chromosomes in some species (1500 species – maize)

- usually no protein-coding genes

- usually negatively affect fitness (fertility)

- not present in all individuals in population

- parazitic DNA (?)

Chromosome number and genome size

TelomeresDNA-protein structures serving to maintain stability of chromosomal ends

Repetitive sequences synthetized by telomerase after replication(TTTAGGG)n in Arabidopsis

Some plants have typical mammalian sequence: (TTAGGG)n

Telomerase - RT with an RNA template

repeat number depends on:- species- developmental stage- cell type - chromosome (within a cell)

Keeping telomere length

- attachement of chromatids- defined by presence of histone CenH3 - CenH3 – kinetochore – spindel fibers

Types:- point (125 bp, yeast Saccharomyces) - holocentric chromosomes (CenH3 along whole chr.)

e.g. Luzula – allows fragmentation- classical – region of different length formed

with heterochromatin (repeats, TE)= epigenetically defined

(neocentromeres)- various strenth in hybrids

Centromeres

Chromosomes: ((telocentric, acrocentric, metacentric,submetacentric))

Crossing of WT Arabidopsis with a line carrying modified (weaker) CenH3 issues in haploid progeny

– inefficient segregation of chromosomes (elimination)

Ravi and Chan 2010

Rabl’s arrangement of chromosomes in interphase nuclei

centromeres and telomeres localized in oposite sites(chrom. size above 500 Mb)

WHY?

Chromosomal territoriesRegions in nucleus occupied with certain chromosome(postmitotic decondensation 2 hours, 2.5 fold increse)

Cremer and Cremer 2010

Experimental confirmation of chromosomal territories

- laser injury, detection of reparation

- specific labelling of chromosomes (FISH)

Sex chromosomes in plants

Sexuality in various taxons of plants evolved independently and repeatedly (5 % species, in about 75 % plant families)

- Marchantia, Gingo, Silene, Rumex, Hop, Poplar …

- sex determination (single locus or more loci)

Sex chromosomes in plantsMorphological classification of sex chromosomes- homomorphic

- heteromorphic

- polymorphic – more than two types:

e.g. Rumex acetosa: male XY1Y2, female XX

Humulus lupulus var. cordiflorus: male X1X2Y1Y2, female X1X1X2X2

Evolution of sex chromosomesFormation of sex chromosome and single sex individuals

– primary mutations causing male and female sterility in loci in strong genetic linkage (intermediarily usually gynodioecy)

model: female (XX) – an allele (in locus A) necessary for development of male sex organs is non-functional in X-chromosome ancestor

(recessive allele)

male XY – an allele (in locus B, linked with locus A) is mutated to suppress formation of female sex organs (dominant allele), this allele is linked with functional allele in locus A

Evolutionary young – homomorphic (recombination only partially limited)

Degenerations (inversions, TE amplification, deletions) - heteromorphic

Splitting or translocations can issue in polymorhic

Polyploidy complicates formation of sex chromosomes