Genetic analysis of the diabetic GK rat has revealed several diabetes susceptibility loci. Congenic strains have been established for the major diabetes locus, Niddm1, by transfer of GK alleles onto the genome of the normoglycemic F344 rat. Niddm1 was dissected into two subloci, physically separated in the congenic strains Niddm1b and Niddm1i, each with at least one disease susceptibility gene. Here we have mapped Niddm1b to 1 cM by genetic and pathophysiological characterization of new congenic substrains for the locus. The gene encoding insulin-degrading enzyme (IDE:) was located to this 1 cM region, and the two amino acid substitutions (H18R and A890V) identified in the GK allele reduced insulin-degrading activity by 31% in transfected cells. However, when the H18R and A890V variants were studied separately, no effects were observed, demonstrating a synergistic effect of the two variants on insulin degradation. No effect on insulin degradation was observed in cell lysates, indicating that the effect is coupled to receptor-mediated internalization of insulin. Congenic rats with the IDE: GK allele displayed post-prandial hyperglycemia, reduced lipogenesis in fat cells, blunted insulin-stimulated glucose transmembrane uptake and reduced insulin degradation in isolated muscle. Analysis of additional rat strains demonstrated that the dysfunctional IDE: allele was unique to GK. These data point to an important role for IDE: in the diabetic phenotype in GK.

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.

Opioid receptors are critical for heroin dependence, and A118G SNP of the opioid receptor gene (OPRM1) has been linked with heroin abuse. In our population of European Caucasians (n ‫؍‬ 118), Ϸ90% of 118G allelic carriers were heroin users. Postmortem brain analyses showed the OPRM1 genotype associated with transcription, translation, and processing of the human striatal opioid neuropeptide system. Whereas down-regulation of preproenkephalin and preprodynorphin genes was evident in all heroin users, the effects were exaggerated in 118G subjects and were most prominent for preproenkephalin in the nucleus accumbens shell. Reduced opioid neuropeptide transcription was accompanied by increased dynorphin and enkephalin peptide concentrations exclusively in 118G heroin subjects, suggesting that the peptide processing is associated with the OPRM1 genotype. Abnormal gene expression related to peptide convertase and ubiquitin͞proteosome regulation was also evident in heroin users. Taken together, alterations in opioid neuropeptide systems might underlie enhanced opiate abuse vulnerability apparent in 118G individuals.drug abuse ͉ dynorphin ͉ enkephalin ͉ mRNA

Autoantibodies against 21-hydroxylase (P450c21) are common in idiopathic autoimmune Addison's disease. In the present work, we have developed a sensitive radiobinding assay using in vitro translated recombinant human 35S-P450c21. Levels of P450c21 antibodies (P450c21-Ab) were expressed as a relative index (P450c21 index) using a P450c21-Ab positive Addisonian serum and two antibody-negative healthy sera as positive and negative standards in healthy individuals. The upper level of normal was the mean + 3 SD. Positivity for P450c21-Ab was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of immunoprecipitated 35S-P450c21. In 38 Addisonian patients, P450c21-Ab were found in 24/28 (86%) idiopathic, 0/5 post-tuberculosis, 0/3 adrenoleukodystrophy, and 0/2 post-adrenalectomy sera. Among healthy individuals, 1/70 (1.4%) were positive. The P450c21 index, as an estimate of P450c21-Ab levels, correlated inversely with the duration of idiopathic Addison's disease (r = -0.527; P = 0.007): 16/16 (100%) positive in patients with less than 20 yr and 8/12 (67%) positive in patients with more than 20 yr disease duration. The availability of this simple and sensitive radiobinding assay to evaluate levels of P450c21-Ab will permit large clinical studies as well as screening subjects at risk. In addition, the general population can now be screened to evaluate the predictive value of P450c21-Ab for Addison's disease.

Striatal enkephalin and dynorphin opioid systems mediate reward and negative affect, respectively, relevant to addiction disorders. We examined polymorphisms of proenkephalin (PENK) and prodynorphin (PDYN) genes in relation to heroin abuse and gene expression in the human striatum and the relevance of genetic dopaminergic tone, critical for drug reward and striatal function. Heroin abuse was significantly associated with PENK polymorphic 3 UTR dinucleotide (CA) repeats; 79% of subjects homozygous for the 79-bp allele were heroin abusers. Such individuals tended to express higher PENK mRNA than the 81-bp homozygotes, but PENK levels within the nucleus accumbens (NAc) shell were most strongly correlated to catecholamine-O-methyltransferase (COMT) genotype. Control Met/ Met individuals expressed lower PENK mRNA than Val carriers, a pattern reversed in heroin users. Up-regulation of NAc PENK in Met/Met heroin abusers was accompanied by impaired tyrosine hydroxylase (TH) mRNA expression in mesolimbic dopamine neurons. In contrast to PENK, no association was detected between PDYN genotype (68-bp repeat element containing one to four copies of AP-1 binding sites in the promoter region) and heroin abuse, although there was a clear functional association with striatal PDYN mRNA expression: an increased number of inducible repeats (three and four) correlated with higher PDYN levels than adult or fetal subjects with noninducible (one and two) alleles. Moreover, PDYN expression was not related to COMT genotype. Altogether, the data suggest that dysfunction of the opioid reward system is significantly linked to opiate abuse vulnerability and that heroin use alters the apparent influence of heritable dopamine tone on mesolimbic PENK and TH function.catecholamine-O-methyltransferase ͉ drug abuse ͉ mu opioid receptor ͉ nucleus accumbens ͉ prodynorphin

Transcription from multiple promoters along with alternative mRNA splicing constitutes the basis for cell-specific gene expression and mRNA and protein diversity. The prodynorphin gene (PDYN) gives rise to prodynorphin (PDYN), precursor to dynorphin opioid peptides that regulate diverse physiological functions and are implicated in various neuropsychiatric disorders. Here, we characterized PDYN transcripts and proteins in the adult human brain and studied PDYN processing and intracellular localization in model cell lines. Seven PDYN mRNAs were identified in the human brain; two of the transcripts, FL1 and FL2, encode the full-length PDYN. The dominant, FL1 transcript shows high expression in limbic-related structures such as the nucleus accumbens and amygdala. The second, FL2 transcript is only expressed in few brain structures such as the claustrum and hypothalamus. FL-PDYN was identified for the first time in the brain as the dominant PDYN protein product. Three novel PDYNs expressed from spliced or truncated PDYN transcripts either lack a central segment but are still processed into dynorphins, or are translated into N-terminally truncated proteins. One truncated PDYN is located in the cell nucleus, suggesting a novel nonopioid function for this protein. The complexity of PDYN expression and diversity of its protein products may be relevant for diverse levels of plasticity in adaptive responses for the dynorphin system.

SUMMARYThe diagnostic specificity of recombinant 21-hydroxylase autoantibodies (21OH-Ab) for Addison's disease was tested in adult patients with either Graves' disease (GD), insulin-dependent diabetes mellitus (IDDM), or polyendocrinopathy, as well as in healthy controls. Using a radiobinding assay with in vitro translated recombinant human 21-hydroxylase, we found 21OH-Ab in 24/28 (86%) idiopathic Addison patients, and using an immunofluorescence assay we found adrenal cortex autoantibodies (ACA) in 12/28 (43%) patients (P ¼ 0·002). All the 12 ACA-positive sera were also positive for 21OH-Ab and ACA were found in 11/15 (73%) patients with less than 15 years and in 1/13 (8%) patients with 15-38 years of disease duration (P ¼ 0·002). 21OH-Ab were present in 3/92 (3%) patients with GD, in 1/180 (0 . 6%) with IDDM and in 0/106 healthy subjects. The 21OH-Ab-positive GD and IDDM patients were also positive for ACA. None of 17 patients with polyendocrinopathy, but without Addison's disease, had 21OH-Ab. None of the 180 Belgian IDDM patients had Addison's disease or developed an adrenal insufficiency at follow up. In two out of three Graves patients, the presence of 21OH-Ab was associated with clinical and biochemical signs of adrenal insufficiency. Of the 89 21OH-Ab-negative patients with GD none had Addison's disease at the time of blood sampling, and 79 were followed up for 5 . 6-7 . 5 years and none developed clinical signs of adrenal insufficiency. We conclude that the presence of 21OH-Ab in patients with endocrine autoimmune diseases is highly specific for Addison's disease.

Lesions in the gene encoding steroid 21-hydroxylase result in congenital adrenal hyperplasia, with impaired secretion of cortisol and aldosterone from the adrenal cortex and overproduction of androgens. Mild forms of the disease cause late-onset symptoms of hyperandrogenism and are thought to be largely underdiagnosed. A limited number of mutations account for the majority of mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. We previously reported a rare allele in two siblings with late-onset disease. This allele contained three sequence alterations, a C to T transition 4 bases upstream of translation initiation, a CCG to CTG change at codon 105 (P105L), and a CCC to TCC transition at codon 453 (P453S). The latter mutation has been found in other ethnic groups, whereas P105L seems to be unique to this family. We have now analyzed the functional consequences of the -4, P105L, and P453S sequence alterations by in vitro translation and after expression of mutant enzyme in cultured cells. As expected, the base substitution at position-4 had no measurable effect on gene expression. The P105L mutation reduced enzyme activity to 62% for 17-hydroxyprogesterone and 64% for progesterone, and the P453S mutation reduced activity to 68% and 46%, respectively. When present in combination, the two mutations caused a reduction of enzyme activity to 10% for 17-hydroxyprogesterone and 7% for progesterone. These results indicate that P105L and P453S can be expected to result in a very subtle disease manifestation when not found in combination, motivating their inclusion when genotyping to ascertain undiagnosed patients with the mildest forms of 21-hydroxylase deficiency.