Human gene expression and genomic imprinting

1. Human gene expression and genomic imprinting Lecture 3

2. PROMOTERS – are combinations of short sequence elements (usually located in the immediate upstream region of the gene- often within 200 bp of the transcription start site) which serve to initiate transcription.Position of cis-acting elements within promoter sequences • TATA box, usually found at a position about 25 bp upstream (-25) from the transcriptional start; it is typically found in genes which are actively transcribed by RNA pol II • GC box found in a variety of housekeeping genes, it appears to function in either orientation • CAAT box often located at position -80; it is usually the strongest determinant of promoter efficiency

3. Basal transcription apparatus • Various general transcription factors (TF) are required to bind to a promoter sequences in the immediate vicinity of a gene, in order to subsequently position and guide the RNA polymerase that will transcribe the gene

4. Basic terms Sense strand – the DNA strand of a gene that is complementary in sequence to the template (antisense) strand, and identical to the transcribed RNA sequence except (that DNA contains T where RNA has U). Template strand – in transcription , the DNA strand that base pairs with nascent RNA transcript Quoted gene sequences are always of the sense strand, in the 5´ 3´direction

5. Cis-acting gene sequences -specific recognition elements recognized by tissue-specific TFs • ENHANCERS – positive transcriptional control elements which are particularly prevalent in mammals; they serve to increase the basal level of transcription which is initiated through the core promoter elements • They function is independent of both their orientation and the distance (in some extent) • SILENCERS – serve to reduce transcription levels; • RESPONSE ELEMENTS – modulate transcription in response to specific external stimuli; they are usually located upstream of the promoter element (often within 1 kb of the transcription start site) • A variety of such elements respond to the specific hormones (e.g. retinoic acid or steroid hormones such glucocorticoids)

7. Chromatin conformation: DNA methylation and the histone code

8. Nucleosome – structural unit of chromatin; it consists of a central core of eight histone proteins (2x H2A,H2B, H3 a H4) around which a strech of 146 bp of dsDNA is coiled; adjacent nucleotides are connected by a short length of spacer DNA • The strings of beads, approx. 10 nm in diameter, are in turn coiled into a chromatin fiber ; the interphase chromosomes seems to consists of these chromatin fibers

9. Histone code • A common shorthand is used for histone modifications. Specific aminoacid residues are identified by the type of histone, the one-letter amino-acid code and the position of the residue counting from N terminus. Thus, H3K9 lysine is lysine 9 in histone H3

10. Histone modification • Histone acetylation • Histone acetyltransferases (HATs) (K=lysine); reduced affinity between histones and DNA ; transcriptional co- activators • Histone methyltranseferases (HMTs) • Histone deacetylases (HDACs); promoting transcriptional repression ; are recruited as part of corepressor complex in response to the DNA methylation

11. Enzymes responsible for DNA methylation • DNA methyltransferases DNMT DNMT1 • Methzlation is sex-specific regulated - pohlavne špecifická regulácia Dnmt1 metyltransferázy • 1so somatic • 1sp sperm • 1oo egg

12. DNA methylation is accomplished by DNA methyltrasnferases at CpG islands • Genomic regions with high frequency of CpG dinucleotides; CpG islands are typically 300 – 3 000 bp in length The usual formal definition of a CpG island is a region with at least 500 bp and with a GC percentage that is greater than 55%

13. Methyl-CpG binding proteins with methyl-CpG-binding domain (MBD) • MEPC2 on X chromosome – loss of function mutations in MEPC2 is responsible for dominantly inherited Rett syndrome Normal delivery, heterozygous girls develop normally for their first year but the regress Other main criteria include loss of purposeful hand skills, loss of spoken language, gait abnormalities, and stereotypic hand movements. 80-90% - dominant de novo germline loss of function mutations (from fathers germline) in MECP2 na Xq28 MECP2 is a transcription factor – methyl-CpG binding protein

14. Epigenetic mechanisms of gene control describes heritable states which do not depend on DNA sequence • (Genetic mechanisms explain heritable states (characters) which result from changes in DNA sequences (mutations)) • DNA methylation Gene repression • (Host defense against transposons or foreign DNA)

15. Epigenetic mechanism and long range control of gene expression • Epigenetic changes- are heritable, but that do not depend on change in genome sequence • DNA methylation – general method of maintaining repression of transcription • - involved in mechanism operating on some genes to ensure that only one of the two parentally expressed alleles is normally expressed (monoallelic expression), even although the nucleotide sequence of the nonexpressed allele may be identicalto that of the expressed • X-chromosome inactivation • Genomic impriting

16. Genomic imprinting • „Imprint“ - nonequivalence in expression of alleles at certain gene loci dependent on the parent of origin • Only one parental allele is expressed • Epigenetic process (cause – parentally specific DNA methylation

17. Genomic imprinting- nonequivalence in expression of alleles at certain gene loci dependent on the parent of origin • Uniparental disomyis pathogenic Some conceptuses have normal 46,XX or 46, XY karyotype but may have inherited two copies of the same chromosome from just one of the parents . This may result in abnormal phenotypes which are different according to parental origin of the relevant chromosome.

18. Angelman syndrome (OMIM 105830) mental retardation, lack of speech, growth retardation, hyperactivity, spasticity inappropriate laughter Prader-Willi syndrome Mental retardation hypotonia, gross obesity, excessive and indiscriminate eating habits, male hypogenitalism small hands and feets

19. In approximately 70 percent of cases there is cytogenetic deletion involving the proximal long arm of chromosome 15q11-13 the same picture for AS and PWS • The same deletions – different syndromes Deletion of certain chromosomal regions produces a different phenotype when on the maternal or paternal chromosome.

20. Subchromosomal mutations causing differential abnormal phenotypes according to parent of origin • Deletion of certain chromosomal regions produces a different phenotype when on the maternal or paternal chromosome. • The same deletions – different syndromes Prader-Willi syndrome – deletion 15q12 on the paternal chromosome Angelmano syndrome - deletion 15q12 on the maternal chromosome

21. Subchromosomal mutations causing differential abnormal phenotypes according to parent of origin • Deletion of certain chromosomal regions produces a different phenotype when on the maternal or paternal chromosome. • The same deletions – different syndromes Prader-Willi syndrome – deletion 15q12 on the paternal chromosome Angelmano syndrome - deletion 15q12 on the maternal chromosome 5q12 IS AN IMPRINTED REGION Chromosomal mutations: De novodeletion4-4,5Mb; deletion on paternal chr. 15 causes PWS, on maternal AS Uniparental disomy – both homologues of a particular pair (chr. 15) are inherited from one parent.; Maternal UPD - lacking the paternal 15, the person develop PWS PAternal UPD – lacking the maternal 15, AS

22. Pathogenic gene expression Genetic changes in the regulatory mechanism of the control elements of gene expression – examples • 1. mutations within the promoter region • 2. mutation within enhancers, silencers and response elements • 3.non-physiological gene expression – control of inappropriate enhancer, silencer or responseelements e.g. gene translocation • 4. mutations in conserved splicing sequences DETECTION of PATHOLOGICAL GENE EXPRESSION – diagnostic, prognosic and predictive consequences

23. Aberrant expression of repressor is sometimes associated with neoplasia. The t(15;17) chromosomal translocation that occurs in promyelocytic leukemia fuses the PML gene to a portion of the retinoid acid receptor (RAR). This event causes unregulated transcriptional repression in a manner that precludes normal cellular differentiation. The addition of the RAR ligand, retinoic acid, activates the receptor, allows cells to differentiate and ultimately undergo apoptosis. This mechanism has therapeutic importance as the addition of retinoic acid to treatment regimens induces a higher remission ate in patients with PML.

24. Proto-oncogenes activation in lymphoma and leukemia • An intact gene is translocated near to a regulatory domain of an other gene, which is for this particularly cell type specific (e.g. IgH) and which is highly expressed within the cell. • Chimerical gene fusion which leads to a production of fusion transcripts and proteins, such fusion protein can geta new pathological function.

25. T(8;14) Burkitt lymphoma . • MYC comes under controll of Ig genes locus control region (similar like enhancer) _high expression of transcirption factor MYC – activation of genes Quantification of c-myc expression

26. Activation by translocation that creates a novel chimeric gene • This mechanism is rare in carcinomas, but common in hematologic tumors and sarcoma. • The best known example is the Philadelphia (Ph) chromosome, a small acrocentric chromosome seen in 90% of patients with chronic myeloid leukemia. • This chromosome is one product of a balanced reciprocal í;22 translocation. The breakpoint on chromosome 9 is within an intron of the ABL oncogene. The translocation joins the 3´part of the ABL genomic sequence onto 5´part of the BCR (breakpoint cluster region( gene on chromosome 22, creating a novel fusion gene. • This chimeric gene is expressed to produce a tyrosine kinase related to ABL product but with abnormal transforming properties.

27. Methods of mRNA analysis • Nothern Blot • Reverse Transcription PCR • Real Time PCR • Microarrays