chapter  5
42 Pages

GENE SILENCING

Cassette Exchange ........................................................................119 5.2.2 Recombinase-Mediated Cassette Exchange, Transfection,

and Transgene Selection ...............................................................119 5.2.3 siRNA-Mediated Knockdown ........................................... 120 5.2.4 Preparation of Genomic DNA, Bisulphite Treatment, T/A

Cloning, and Sequencing ............................................................. 120 5.2.5 Native Chromatin Immunoprecipitation Assay and

Quantitative PCR ......................................................................... 121 5.2.6 RNA Isolation, Reverse Transcription and Quantitative

RT-PCR ........................................................................................ 122 5.2.7 Whole-Cell Protein Extracts and Western Blot Analysis ... 122 5.2.8 Northern Blot Analysis ...................................................... 123 5.2.9 Fluorescence-Activated Cell Sorting and Analysis of

Cassette Integration ...................................................................... 123 5.2.10 H3K9me3 Profiling of Endogenous Retroviruses ........... 123

5.2.11 H3K9me3 Profiling in the Sequences Flanking Endogenous Retroviruses ............................................................ 124

5.3 Discussion .................................................................................... 125 5.3.1 Role of H3K9me3 and HP1 in Silencing of ERVs ............ 125 5.3.2 H3K9me3-Dependent, H3K9me3 Reader-Independent

Proviral Silencing? ....................................................................... 125 5.3.3 Heterochromatin Spreading into Sequences

Flanking ERV ............................................................................... 127 5.4 Results .......................................................................................... 128 5.4.1 Catalytic Activity of SETDB1 is Largely Required for

ERV Silencing .............................................................................. 128 5.4.2 Depletion of HP1β but not HP1α Leads to Modest

Upregulation of Select ERV Families .......................................... 130 5.4.3 Depletion of HP1α Results in a Modest Reduction of

DNA Methylation at IAPEz ERVs ............................................... 132 5.4.4 Neither HP1α nor HP1β are Essential for H4K20me3

Deposition at ERVs ...................................................................... 132 5.4.5 HP1β plays a Role in the Spreading of H4K20me3 but

not H3K9me3 from ERVs into Flanking Genomic Regions ....... 134 5.4.6 Application of Novel ERV Reporter Lines in a

siRNA-Based Screen of H3K9me3-Binding Proteins ................. 137 5.5 Conclusion ................................................................................... 143 Keywords .............................................................................................. 144 Acknowledgments ................................................................................. 144 Authors’ Contributions .......................................................................... 145 References ............................................................................................. 145 Credits ................................................................................................... 154

5.1 INTRODUCTION

Endogenous retroviral sequences (ERVs) are relics of ancient retroviral integration into the germline. These parasitic elements are abundant in mammals, occupying approximately 8% of the mouse genome and 10% of the human genome [1, 2]. ERVs are subdivided into three diverse classes based on the similarity of their reverse transcriptase genes or their relationship to different genera of exogenous retroviruses. In the mouse, class I ERVs, similar to gammaretroviruses, include active families such as murine leukaemia viruses (MLVs) and murine retroviruses that use tRNAGln (GLN). Class II ERVs are similar to alpha-and betaretroviruses and include Mus musculus ERV using tRNALys type 10C (MMERVK10C), the highly retrotranspositionally active intracisternal A-type particles (IAPEz) and early transposon/Mus musculus type D retrovirus (ETn/MusD) families. Class III ERVs, the oldest and most abundant ERVs, are most similar to spumaviruses and are represented by mouse endogenous retrovirus type L (MERV-L) and mouse apparent LTR retrotransposons (MaLR) [3, 4]. Numerous regulatory motifs in the ERV long terminal repeats (LTRs) can initiate high levels of transcription in tissues and cell lines [5], and there is extensive evidence of aberrant ERV-driven gene expression in cancers [6-11] and tissues of aging mice [12, 13]. In an effort to counteract the potentially detrimental effects of ERVs, eukaryotic genomes have evolved multiple lines of defence against active exogenous and endogenous retroviruses [14], including DNA methylation and repressive histone modifications.