Comprehensive knowledge of immunoglobulin genetics is required to advance our understanding of B cell biology. Validated immunoglobulin variable (V) gene databases are close to completion only for human and mouse. We present a novel computational approach, IgDiscover, that identifies germline V genes from expressed repertoires to a specificity of 100%. IgDiscover uses a cluster identification process to produce candidate sequences that, once filtered, results in individualized germline V gene databases. IgDiscover was tested in multiple species, validated by genomic cloning and cross library comparisons and produces comprehensive gene databases even where limited genomic sequence is available. IgDiscover analysis of the allelic content of the Indian and Chinese-origin rhesus macaques reveals high levels of immunoglobulin gene diversity in this species. Further, we describe a novel human IGHV3-21 allele and confirm significant gene differences between Balb/c and C57BL6 mouse strains, demonstrating the power of IgDiscover as a germline V gene discovery tool.

INTRODUCTION 1.1 EPIGENETIC REGULATION 1.2 CHROMATIN CROSSTALK IN 3D 1.2.1 The innovation of techniques to explore the 3D genome 1.2.2 The genome organizer CTCF and its PARP1 partner 1.2.3 Compartmentalization of nuclear functions in 3D 1.2.4 The active compartments: nuclear interior 1.2.5 The inactive nuclear compartments 1.3 NUCLEOPORINS AND THE GENE GATING PRINCIPLE 1.4 REGULATION OF CIRCADIAN TRANSCRIPTION IN THE COMPARTMENTALIZED NUCLEUS 1.4.1 The central and peripheral clocks 1.4.2 The entrainment of circadian rhythm by external time cues 1.4.3 The clock machinery: driving circadian transcription 1.5 CIRCADIAN CHROMATIN TRANSITIONS 1.5.1 The establishment of active chromatin states by the positive limb 1.5.2 The establishment of repressed chromatin states by the negative limb 1.5.3 Crosstalk between the positive and negative limb of the clock machinery during chromatin transitions 1.6 CIRCADIAN CLOCK, CELLULAR METABOLISM AND COMPLEX DISEASES 2 AIMS 3 METHODS AND MATERIALS 3.1 CELL CULTURES AND TREATMENTS 3.2 RNA/DNA FISH ANALYSES 3.3 IN SITU PROXIMITY LIGATION ASSAY (ISPLA) 3.4 CHROMATIN IN SITU PROXIMITY (CHRISP) 3.5 GRID WIDE-FIELD MICROSCOPY 3.6 CHROMATIN NETWORKS AND INTEGRATION ANALYSES 3.6.1 Circular chromatin conformation capture sequencing (4C-Seq) 3.6.2 Nodewalk 3.7 RNA ANLYSES 3.7.1 Pulse labeling of RNA 3.7.2 The nuclear RNA export assay 3.7.3 mRNA decay analyses 3.7.4 RT-QPCR analysis of transcription 4 RESULTS 4.1 PAPER I: PARP1-AND CTCF-MEDIATED INTERACTIONS BETWEEN ACTIVE AND REPRESSED CHROMATIN AT THE LAMINA PROMOTE OSCILLATING TRANSCRIPTION 4.1.1 Interactome connecting circadian loci and LADs 4.1.2 Molecular ties connecting circadian loci to LADs 4.1.3 The role of the nuclear periphery in circadian transcriptional attenuation 4.1.4 Summary: novel principles in the entrainment of circadian transcription 6 4.2 PAPER II: WNT SIGNALING AND AHCTF1 PROMOTE ONCOGENIC MYC EXPRESSION THROUGH SUPER-ENHANCER-MEDIATED GENE GATING 4.2.1 Regulation of MYC transcription in 3D 4.2.2 Contribution of gene gating to MYC mRNA accumulation 4.2.3 The role of WNT in the super-enhancer mediated gene gating of MYC 5 DISCUSSIONS 5.1 THE ROLE OF NUCLEAR PERIPHERY IN THE REGULATION OF GENE EXPRESSION 5.2 MYC AND THE CIRCADIAN CLOCK 5.3 ADAPTATION TO THE ENVIRONMENT 5.3.1 WNT signaling 5.3.2 Circadian entrainment 5.3.3 Nucleoporins and the transcriptional memory 5.4 CTCF AND PARP1 IN COMPLEX DISEASES 5.4.

Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity.