Gene Expression DNA RNA. Protein. Metabolites, stress, environment

Transcription

1 Gene Expression DNA RNA Protein Metabolites, stress, environment 1

2 EPIGENETICS The study of alterations in gene function that cannot be explained by changes in DNA sequence. Epigenetic gene regulatory mechanisms include: chromatin (heterochromatin, remodeling, histone modifications), DNA methylation, RNA interference. 2

3 Epigenetic modifications are heritable, but reversible, changes associated with chromatin that affect gene expression without alteration of the DNA sequence. Epigenetics gene activation or silencing, extends the information potential of DNA. Epigenomics characterization of the set of epigenetic modifications associated with an entire genome.

4 Important concepts behind the study of epigenetics/epigenomics How does a fixed DNA blueprint allow flexibility in managing changes to environmental signals? Environmental inputs such as nutrition can modulate cell metabolism Sassone-Corsi Science 2013 External (infections, chemical agents & drugs) and internal (cytokines, hormones) environmental stimuli can modify the epigenetic profile of a gene, directly influencing its expression and, ultimately, the cell type and immune response. The epigenome connects the genome with the cellular environment and determines cellular identity and functionality. Suarez-Alvarez et al Epigenetics 2013

5 The ability to package large genomes is associated with a fundamental shift in the logic of gene regulation between prokaryotes and eukaryotes. In prokaryotes, the ground state is non-restrictive; in eukaryotes, transcriptional activity is generally impeded by nucleosomal packaging. Activators and repressors influence gene expression by recruiting chromatin modifying activities to promoters. Richards and Elgin 2002.

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7 Jenuwein, T., David Allis, CD. Science 293: 1074,

8 Komberg, RD. Cell 98: ,1999 8

9 ATP-dependent chromatin remodeling complexes Protein complexes that use ATP hydrolysis to alter the physical structure of chromatin by loosening nucleosomal DNA, sliding/repositioning nucleosomes, ejecting or inserting variant histones. Different classes of complexes function in forming nucleosomes during replication or local remodeling of chromatin for transcription initiation and elongation.

10 ATP-dependent multiprotein chromatin remodeling complexes About 12 multiprotein complexes [5-20 subunits] falling into 4 groups of complexes depending on ATPase subunit: 1) SWI/SNF SNF, BAF, BRG/BRM 2) ISWI - ISW1/2, RSF, NURF, CHRAC, ACF 3) NURD/Mi-2/CHD 4) INO80

11 Four ATPase subfamilies Becker & Workman CSH Perspectives in Biology

12 Regulation by remodeling complexes Different transcriptional activators may recruit different complexes Subsets of genes may be affected by specific modifying complexes Alternative histone incorporation (e.g. H2A.Z, H2A.X, H3.3, etc) A spectrum of amino acids may be modified to recruit specific readers and determine outcome. Chromatin remodelers are required during transcription for initiation, elongation and repression.

13 Maze et al (2014) 15:

14 Epigenetic repression of gene expression The Chromatin Group, Department of Anatomy, The Medical School, Birmingham, B15 2TT, U.K.

15 Lewin, Benjamin Genes VII. Oxford University Press Inc., New York: 582,

16 The histone code Distinct histone amino-terminal modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins which in turn dictate dynamic transitions between transcriptionally active or silent chromatin states. The combinatorial nature of histone amino-terminal modifications thus reveals a histone code that considerably extends the information potential of the genetic code. Jenuwein and Allis. Science 2001, 293: We commonly refer to readers, writers and erasers of epigenetic marks.

17 Cheung, P. et. al., Cell, Vol. 103, p , October 13,

18 Epigenetic marks on histones Ubiquitination (lysine) ubiquitination deubiquitination Acetylation (lysine) HDACs HATs Nucleosome Histones HMTs (MLL, Set, Dot, PRMT) HDMs (LSD, JMJ) Methylation (lysine, arginine) Kinases Phosphatases Phosphorylation (serine, threonine, tyrosine, histidine) 18

19 Histone acetylation Induces new DNase I hypersensitivity sites within the nucleosome by opening the chromatin structure Enhances binding of transcription factors Activates transcription of certain genes Nucleosome incorporation of unacetylated histones is repressive Reduces capacity of histone HI to compact chromatin Transcriptionally active chromatin [euchromatin] manifests enhanced acetylation compared to inactive chromatin [heterochromatin] Activates integrated retroviral sequences HIV Can compete or synergize with other modifications

20 Different HATs display specificity for histones and residues: GCN5 - H3 [K 14 ] CBP/p300 - H3 [K 14 and K 18 ] and H4 [K 5 and K 8 ] PCAF - H3 [K 14 ] and H4 [K 8 ] Mizzen, C.A. et al., Cell Mol Life Sci 54:6,

21 Histone deacetylation There are families of HDACs with different specificities I HDAC1,2,3,8 IIA HDAC4,5,7,9 IIB HDAC6,10 III SIRT1-7 IV HDAC11 These deacetylases are recruited to various repressive complexes e.g. NCoR/SMRT/HDAC3, Sin3a/b/HDAC1/2. Differential substrate usage, subcellular localization and expression contribute to the selective function of deacetylases. HDAC inhibitors are in clinical use.

22 DNA methylation Mostly at CpG dinucleotides Roles in X chromosome inactivation, genomic imprinting, retrotransposon silencing Disruptions in methylation or readers associated with pathology MBD readers (MBD and MeCP2 proteins), Zn finger readers (SRA -- SET and RING assoc domain -- proteins) erasers TET oxidation, UDG BER 22

23 Mechanisms of DNA methylation

24 Persistence of epigenetic states Maintenance of repressed state by methylation Asymmetrically methylated CpG segregates one CH 3 group to each daughter chromatid resulting in hemi-methylated DNA Dnmt-1 [maintenance DNA methyltransferase] binds to the replication fork and completes methylation CpG binding proteins [MeCP2] bind methylated CpGs and recruit HDAC to reestablish repression in each daughter cell

25 Transcriptional suppression by methylation of CpG islands Direct block to transcription factor/complex binding at promoter sites Some sequence specific TFs are methylation dependent (KLF) Methylated cytosine binding proteins recruit HDACs Compaction of higher order chromatin

26 DNA methylation in normal and cancer cells DNA is generally found to be hypomethylated in tumors BUT CpG islands in gene promoters are frequently hypermethylated. Baylin,S. Nature Clin Practice Oncology 2:S4-S11,

27 Epigenetic alterations in tumor progression Esteller M. N Engl J Med 2008;358:

28 Epigenetics in cancer management Esteller M. N Engl J Med 2008;358:

29 Epigenetic inactivation of tumor-suppressor genes Esteller M. N Engl J Med 2008;358:

30 Epigenetic regulation of immune responses DNA methylation and demethylation are represented by black and white lollipops, respectively; histone modifications are shown as circles: green, H3K4me3; red, H3K27me3; purple, H3K9me3; blue, acetylation of H3 or H4 Suarez-Alvarez et al Epigenetics

31 Epigenetic regulation CD4+ T cell differentiation Suarez-Alvarez et al Epigenetics