Accumulating evidence indicate that molecular mechanisms generating circadian rhythms display some degree of tissue‐specificity. More specifically, distinct patterns of expression for nuclear receptors of the ROR family indicate that the transcriptional control of the clock gene Bmal1 differs among tissues. This study aims to investigate the expression of Rorγisoforms (Rorγ and Rorγt) and characterize the molecular mechanisms underlying their tissue‐specific expression. The expression of Rorγ isoforms was assessed in mouse liver, muscle, thymus and testis throughout 24 h using quantitative RT‐PCR. Although the expression of Rorγ was rhythmic in the liver and thymus, it was constitutively expressed in muscle and testis. In contrast, the expression of Rorγt was constitutive in all four tissues. Furthermore, rhythmic expression of Rorγ was impaired in Clock mutant mice whereas the mutation had no effect on Rorγt expression. In line with these findings, luciferase assays revealed that transcription of the Rorγ promoter is clock‐controlled whereas that of Rorγt promoter is essentially clock‐independent. Our results provide insights into the molecular mechanisms that lead to differential expression of Rorγ and Rorγt and are suggestive of a framework that might account for tissue‐specific circadian regulation.

The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity.

The functioning of the genome is tightly related to its architecture. Therefore, understanding the relationship between different regulatory mechanisms and the organization of chromosomal domains is essential for understanding genome regulation. The majority of imprinted genes are assembled into clusters, share common regulatory elements, and, hence, represent an attractive model for studies of regulation of clusters of non-paralogous genes. Here, we investigated the relationship between genomic imprinting and diurnal regulation of genes from the imprinted domain of mouse chromosome 12. We compared gene expression patterns in C57BL/6 mice and congenic mice that carry the imprinted region from a Mus musculus molossinus strain MOLF/Ei. In the C57BL/6 mice, a putative enhancer/oscillator regulated the expression of only Mico1/Mico1os, whereas in the congenic mice its influence was spread onto Rtl1as, Dio3 and Dio3os, i.e. the distal part of the imprinted domain, resulting in coordinated diurnal variation in expression of five genes. Using additional congenic strains we determined that in C57BL/6 the effect of the putative enhancer/oscillator was attenuated by a linked dominant trans-acting factor located in the distal portion of chromosome 12. Our data demonstrate that (i) in adult organs, mRNA levels of several imprinted genes vary during the day, (ii) genetic variation may remove constraints on the influence of an enhancer and lead to spreading of its effect onto neighboring genes, thereby generating genotype-dependent expression patterns and (iii) different regulatory mechanisms within the same domain act independently and do not seem to interfere with each other.

Most of the known imprinted genes are assembled into clusters that share common imprinting control regions (ICRs).Non-coding transcripts are often associated with ICRs and implicated in imprinting regulation. We undertook a systematic search for transcripts originating from the Dlk1-Gtl2 intergenic region that contains the ICR for the chromosome 12 imprinted cluster and identified two overlapping transcripts expressed from opposite strands exclusively from the maternal chromosome. These novel imprinted transcripts most likely represent non-coding RNAs and are located telomeric to the IG DMR, extending the proximal boundary of the region of maternal-specific transcription. Their expression is tissue-specific and shows diurnal and circadian oscillations. Therefore, we named these novel transcripts maternal intergenic circadian oscillating 1 (Mico1) and Mico1, opposite strand (Mico1os).