Shaun Cowley's Research Interests

The control of gene expression by histone-modifying enzymes

Selected Publications

Introduction

The ability to regulate the transcription of protein coding genes in response to growth factors, cellular stress, DNA damage or normal development is one of the most fundamental processes of life. Our laboratory utilizes transgenic mice, ES cell culture and biochemical approaches to address the function of ‘histone modifying’ enzymes and their associated complexes, in an effort to understand the molecular mechanisms by which they control gene expression in vivo.

Our primary interest is the function of a class of histone modifying enzymes called, histone deacetylases (HDACs). HDACs, as their name implies, remove the acetyl group from lysine residues present within histone tails. HDACs are utilized by a variety of transcriptional repressors, in many cell types, to inhibit transcription at a gene specific level (for a detailed discussion of HDAC enzymes, see below).

In addition to acetylation, we are also studying the effects of histone methylation on the levels of gene activity in vivo. LSD1 (lysine specific demethylase 1) was the first protein demethylase to be isolated and characterized. We are currently analyzing the phenotypic consequences of loss of LSD-1 activity, using LSD1 knock-out mice generated in the lab (see below).

 

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The transcription of protein coding genes is controlled on various levels . The DNA primary sequence: defines the protein coding elements of the genome and contains conserved non-coding elements which are the binding sites for transcription factors. DNA modification: Approximately 3-4% of cytosines are methylated in normal human cells. This generally occurs at the di-nucleotide CpG. Concentrated levels of DNA methylation (hypermethylation) around genes often results in transcriptional silencing. Chromatin: The DNA in each cell is not naked but is entwined with a restrictive coat of histone and non-histone proteins which is collectively termed chromatin. The transcriptional potential of a gene is directly related to its chromatin context, which in turn, is influenced by the covalent modification of its core histone components. Histones form a physical impediment to the cellular machinery which needs access to the DNA in order to transcribe protein coding genes. Post-translational modification of histones is therefore an important method for regulating access to DNA.

 

Transcription and Histone Acetylation –

Acetylation of lysine residues within the N-termini of histones H2A, H2B, H3 and H4 correlates with regions of transcriptional activity. Conversely, histones within heterochromatin, regions which contain few active genes, are generally hypoacetylated. This simple dichotomy is best observed in the process of X chromosome inactivation where the inactivated X is almost absent of acetylated histone H3 and H4, while histones incorporated into the active X are abundantly acetylated [1]. Histone acetylation is a reversible process. Removal of the acetyl moiety from lysine residues in histone tails is catalyzed by a family of enzymes termed ‘histone deacetylases’ (HDACs). Loss of the acetyl group restores the lysine’s positive charge, thus increasing the natural affinity of the histone tail for the phosphate backbone of DNA, creating a repressive chromatin conformation. Transcription factors are able to manipulate this transient modification of chromatin to positively or negatively regulate transcription at a gene specific level. There are 18 HDAC enzymes in mammalian cells which can be broadly classified into three groups: Class-I HDACs (1, 2, 3 and 8) which are widely expressed, Class-II HDACs (4, 5, 6, 7, 9 and 10) that share a similar catalytic domain to class-I enzymes but have larger N-termini and are more restricted in their expression patterns, and the Sirtuin family of NAD dependent HDACs (Class-III).

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Class-I HDACs: repression, proliferation and cancer –

Mammalian Class-I HDACs (1, 2, 3 and 8) are abundant, ubiquitously expressed and share a high degree of sequence similarity. They are located almost exclusively in the nucleus as part of multi-protein complexes which regulate gene expression by modulating chromatin structure (see figure of known complexes, below).

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One of the best characterized examples of a class-I HDAC containing complex, is the mSin3A complex, which contains mSin3A, HDAC1 or 2, Sap18, Sap30, SDS3 and RbAP48 [2]. Often the interaction between HDACs and transcriptional repressors is indirect, mediated through co-repressor proteins, such as mSin3A. Over 40 different DNA bound repressors (including tumour suppressors p53 and Rb) function by interacting with mSin3A 3, thereby increasing the local concentration of HDAC1 (or 2), which deacetylate histones, resulting in a more repressive form of chromatin. One such repressor is the methylated CpG binding protein, MecP2. Mutations in Mecp2 cause Rett Syndrome a neurodegenerative disease that affects predominantly young girls [4]. The mSin3A/HDAC complex is thus an excellent paradigm for the study of HDAC containing complexes.

 

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Collectively, these HDAC containing complexes through a variety of protein-protein interactions, that have yet to be fully characterized, have been implicated in many processes including, cell cycle progression [5], DNA repair [6], differentiation [7,8], genomic stability [9] and cancer [10].

Clinically class-I HDAC enzymes are of great interest because they are ‘druggable’. Inhibition of HDAC activity in cancer cell lines, using small molecule inhibitors, results in cell cycle arrest or apoptosis. Currently, HDAC inhibitors (HDACi) are being tested in clinical trials as anti-cancer agents [11]. In addition, HDACi’s have also been found to ameliorate the neurodegenerative and motor deficits in animal models of Huntingdon’s disease [12,13]. A broad spectrum of possibilities exists for the clinical application of HDACi’s. The search is now on to develop specific HDACi’s that will inactivate individual HDAC enzymes. There is therefore a compelling applied, as well as academic, motivation for the study of Class-I HDACs enzymes.

Using specific HDAC knock-out mice and embryonic stem (ES) cells generated in the lab, we are beginning to address the roles of HDACs 1, 2 and 8 in cell cycle progression, embryonic development, thymocyte development and cancer. By building a comprehensive list of repressors which recruit HDACs in vivo and the genes to which they are directly targeted, we hope to understand the molecular mechanism(s) by which they perform these tasks.

Function of the Lysine Specific Demethylase, LSD-1 in vivo

The lysine specific demethylase 1 (LSD-1) was the first protein demethylase to be isolated and characterized [14]. LSD-1 functions by removing the methyl moieties from di-methylated lys4 on histone H3, a marker that correlates with regions of active transcription. Consistent with removing this positive marker of transcription from chromatin, LSD-1 is a constituent of a co-repressor complex which contains CoREST and HDAC1 proteins [15].

 

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We are currently using the LSD1-KO mice generated in our lab to analyze the role of histone methylation in vivo.

References

[1] Chang, S et al. Frontiers in Bioscience 11, 852-866, 2006

[2] Laherty CD, et al. Cell. 1997 May 2;89(3):349-5

[3] Silverstein et al, Curr Genet. (2005) 47: 1–17

[4] Amir et al, Nat Genet. 1999 Oct;23(2):185-8

[5] Lagger et al, EMBO J. 2002 Jun 3;21(11):2672-81

[6] Scott and Plon, Mol Cell Biol. 2003 Jul;23(13):4522-31

[7] Yamaguchi et al, Development. 2005 Jul;132(13):3027-43

[8] Cunliffe and Casaccia-Bonnefil Mech Dev. 2005 Nov 29

[9] Pothof et al, Genes Dev. 2003 Feb 15;17(4):443-8.

[10] Lin et al, Nature. 1998 Feb 19;391(6669):811-4

[11] Dokmanovic M, Marks PA J Cell Biochem. 2005 Oct 1;96(2):293-304

[12] Hockly et al, PNAS. 2003 Feb 18;100(4):2041-6. 2003 Feb 7

[13] Ferrante et al, J Neurosci. 2003 Oct 15;23(28):9418-27

[14] Shi Y et al, Cell 2004 Dec 29;119(7):941-53.

[15] Hakimi et al, Proc Natl Acad Sci U S A. 2002 May 28;99(11):7420-5

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Redfearn Lecture 2017

To Be Confirmed