Long-range gene regulation

Transcription

1 Long-range gene regulation Short Course in Medical Genetics Melbourne, June 2011

2 Question Why should long-range regulation of gene expression be of interest to Clinical Scientists and Pathologists interested in providing genetic diagnostic testing services? Intrinsically interesting and important area New technologies Array/ NGS are going to reveal novel genomic variants, some of which will be disease-causing as a result of disruption to distant regulatory elements rather than to transcriptional units. We need to be able to deliver on the expectation that we can recognise a disease-causing genomic variant when we see one

3 Regulation of gene expression Labile regulation Moment-to-moment control by transcriptional activators and repressors Control exerted by nuclear concentrations, covalent modifications, subunit associations, etc. Epigenetic regulation Has sufficient stability to be transmitted from parental cell to daughter cells Involves alterations to the structure of chromatin, including covalent modification of DNA and histones

4 Labile gene expression Is controlled by regulatory elements that can be located far away along the chromosome In some instances, regulatory elements may be on other chromosomes

5 Promoters for eukaryotic genes encoding polypeptides CG box CAAT box TATA box B-globin gene promoter region Glucocorticoid receptor gene

6 Assembling the basal transcription apparatus onto the promoter Basal transcription apparatus comprises 27 polypeptides

7 Transcription factors may be either upstream or downstream of the transcription start site

8 Distal regulatory elements Enhancers Silencers Insulators Locus control regions

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11 Fractal globule No knots!

12 Chromosome A hierarchically coiled single DNA molecule Chromosomal loops

13 Enhancers Silencers

14 Chromatin loops

15 Characterising long-range gene regulation In a small but significant number of patients the molecular lesions do not directly disrupt the transcribed region of the gene Instead, demonstrated interference with transcriptional regulation of the gene Clinical cases provided the first indications that long-range effects regulated expression of genes

16 Absence of the iris Aniridia

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18 Aniridia Arises from heterozygous loss-of-function mutations within the PAX6 gene on chromosome 11 Some cases demonstrated absence of a PAX6 mutation Cases with chromosomal translocations with breakpoints up to 125 kb downstream of the last PAX6 exon - Demonstrating the presence of distant control elements

19 PAX6 enhancers Positioned within the intron of a neighbouring unrelated gene (ELP4) Some cases demonstrated absence of a PAX6 mutation

20 Holoprosencephaly Malformation of the forebrain arising from failed or incomplete forebrain division

21 Pre-axial polydactyly

22 Acheiropodia

23 SHH Gene Specifies the signaling protein Sonic Hedgehog, which acts to establish the body plan in embryogenesis SHH loss of function holoprosencephaly SHH regulatory lesions preaxial polydactyly acheiropodia

24 SHH Gene

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27 SHH Gene

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30 Genetic diseases triggered by aberrant gene transcription can occur in several ways - Separation of cis-regulatory elements and promoters by chromosomal translocations or inversions Deletion of long range cis-elements Deleterious mutations within cis-elements Disturbing the normal interactions of promoters and cisacting enhancers through appearance of a new promoter Interfering with local chromatin structure by disturbing antisense transcripts regulating more global chromatin structures

31 ENCODE project National Human Genome Research Institute (NHGRI) Goal: To define all the functional DNA sequence elements in the human genome

32 ENCODE pilot study Studied 44 genomic regions encompassing 30Mb (1% of the human genome) Most genomic regions were transcribed, at least in some cells and at some developmental stage Multiple novel transcripts non-coding some previously unknown exons of known genes extra exons were often kb upstream of TSS (some 200 kb upstream). evidence of transcripts containing exons from more than one gene

33 An example of ENCODE data

34 Array Genomic Scanning (Molecular Karyotyping) Use of arrays to detect constitutional aneuploidy in lieu of cytogenetic analysis of chromosomes - diagnostic assessment of infants and children developmental delay pervasive developmental disorders birth defects - miscarried products of conception and stillborn fetuses - likely utility for classification and staging of cancer - emerging use in prenatal diagnosis

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36 Interpretive assessment of array data Clinical interpretation is heavily weighted by - CNV population frequencies (databases; literature) Gene content gene deserts Empirically determined reporting thresholds based on genomic length - around kb for deletions kb for duplications.

37 Gene desert Sometimes contain highly conserved sequences evolutionary protection from chromosomal rearrangements Should we be considering neighbouring upstream and downstream regions? There are instances where the process of checking beyond array coordinates has yielded clinically relevant information

38 Summary Long-range regulation of gene expression - is interesting! - has growing relevance for clinical scientists and pathologists offering genetic diagnostic testing services

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40 "a rough or fragmented geometric shape that can be split into parts, each of which is a reduced-size copy of the whole Fractal globule No knots!

41 ENCODE study tools Multiple techniques used to identify all functional sequence elements - Intensive transcriptome analysis Concerted efforts to identify both cis-acting regulatory DNA elements and their respective trans-acting regulatory proteins Efforts to define chromatin structure and some of its epigenetic regulation

42 Clinical relevance of long-range gene regulation In a small but significant number of patients the molecular lesions do not directly disrupt the transcribed region of the gene Instead, demonstrated interference with transcriptional regulation of the gene Clinical cases provided the first indications that long-range effects regulated expression of genes Treasure the exceptional case!