Saturday, 22 December 2012

Nuclear Structure: Implications for gene expression mechanisms

A mini-review of MAR-binding proteins
Nuclear matrices or nuclear scaffold were defined by biochemical fraction of nucleus after treatment by with detergent, salt and nucleases. They mainly consist of nonhistone protein, RNA and DNA in eukaryote.

Chromatin is looped into domains by attachment of chromatin fibre to nuclear matrix.

DNA sequences that bind preferentially nuclear matrices are matrix attachment region (MAR) or scaffold associated region (SAR). MARs are 200bp long, AT-rich, contain topoisomerase II consensus sequences and other AT-rich sequence motifs.  They are near cis-acting regulatory sequences. Their binding sites are abundant.

They are enriched in inverted repeats, AT tracts, DNA unwinding elements, replication initiator protein sites, homooligonecleotide repeats, etc.

Many known MARs do not have extensive sequence homology. They appear to be functionally conserved. Animal MARs can bind plant nuclear scaffolds and vice versa.

Role of nuclear organisation in cancer
Nuclear matrix proteins involved in regulating gene expression, DNA replication and repair. Chromatin folding facilitates interactions between genomic regions to enable or repress transcription. It enables individual genomic regions to be insulted from surrounding regions.  Nuclear matrix proteins are aberrantly expressed in cancer.

Nuclear matrix proteins as cancer markers
Proteins aberrantly expressed in spec forms of cancer can be used to improve diagnosis.

Generation of chromosome rearrangements
2 models for translocation in cancer.

In breakage first model, genomic regions containing double stranded breaks move in nucleus. They can illegitimately recombine with other genomic regions with DSBs.

In contact first model, local recombination events arise at DSBs in chromosomes already proximally positioned. Contact-first model appears to be correct.

Gene fusions caused by translocations in cancer cells correlates with frequency of side by side pairing of genes in normal control cells.

Replication and transcription: shaping landscape of genome by Chakalova et al

The proteomes of trascnription factories containing RNA polymerases I, II and III by Melnik et la

(a) Strategy. Cartoon shows a chromatin loop with nucleosomes (green circle) tethered to a polymerizing complex (oval) attached to the substructure (brown). The cells are permeabilized and in some cases a run-on is performed in [32P]UTP so that nascent RNA can be tracked. The nuclei are then washed with NP-40, most of the chromatin is detached with a nuclease (here, DNase I), the chromatin-depleted nuclei are resuspended in NLB and polymerizing complexes are released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and the complexes are partially resolved in two-dimensional (2D) gels (using blue native and native gels in the first and second dimensions, respectively); rough positions of complexes (and a control region, labeled 'C') are shown. Finally, different regions are excised, and their content is analyzed by mass spectrometry. (b) Recovery of [32P]RNA, after including a run-on. Fractions correspond to those at the same level in a. (c) Run-on activity assayed later during fractionation (as in a, but without run-on at beginning). Different fractions, with names as in a, were allowed to extend transcripts by less than 40 nucleotides in [32P]UTP, and the amount of [32P]RNA per cell was determined by scintillation counting. Fractions '2pellet' and '4pellet' were also resuspended in NLB before run-ons were performed; results indicate that NLB reduces incorporation to half or less. Despite this, '5super' has 25% of the run-on activity of permeabilized cells ('2pellet'), which is equivalent to half of the original (after correction for the effects of NLB).

(a) Numbers of proteins in the different complexes and their overlap. (b) Many proteins in each complex are associated with the GO term 'gene expression' (GO: 0010467), and complex II contains more with 'transcription from RNA polymerase II promoter' (GO: 0006366) than do complexes I and III. (c) Most proteins in each complex possess GO terms related to transcript production. Selected GO terms were incorporated into eight groups; for example, 'transcription' includes terms 'RNA polymerase', 'transcription factor' and 'transcription regulation'), and 'other terms' includes those not in the other seven groups. Four additional sets of proteins are included for comparison on the right. Some proteins possess terms in more than one group, and terms in each group are expressed as a fraction of the total in all groups. In each complex, 2% of proteins lacked any GO term, and many proteins in the complexes associated with 'other terms' nevertheless turn out to have a role in transcript production (for example, actin21 proteasomal constituents17 and nucleoporins22). Each complex has a characteristic pattern, which is distinct from those given by proteins with the terms 'cytoplasm' and 'S100'

Replication and transcription: shaping the landscape of the genome

Mouse haemoglobin β-chain complex (Hbb) genes and their distal regulatory elements, locus control region (LCR) more than 50kb away engage and specific higher order, loop structures during transcription.

Only  1 out ot 9 distal regulatory elements clustering around active Hbb gene is known to be a classical enhancer.  Other sequence elements have insulator like properties, intergenic promoter activity. They coalesce into an active chromatin hub.

A hub that is formed by long-range interactions between the haemoglobin beta-chain complex (Hbb) genes and the locus control region is shown. Transcriptional activation involves physical association of genes and their regulatory elements. This arrangement could increase the local concentration of trans-acting transcription factors by spatial clustering of binding sites for such factors95, 96, 105. The coloured circles represent DNaseI hypersensitive sites.
Factories as organisers
Different genes often co-occupy the same factory. This indicates that genes do not assemble their own transcription sites de novo when the become active. They migrate to preassembled transcription site.

Studies indicate that transcription factories assemble on a scaffold or nuclear matrix. A stab;e factory implies that genes of transcription units would be pulled though a factory rather than pols moving along chromatin fibre.

Prokaryotic RNAP is a powerful motor protein. Tractor forces from efficient conversion of free energy due to nucleotide hydrolysis during intergenic transcript elongation could ratchet distal regulatory elements and target genes into a common factory. This facilitates formation of long range cis interactions.

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