Martina Fink

Many facets of mammalian lanosterol 14α-demethylase from the evolutionarily conserved cytochrome P450 family CYP51

Lanosterol 14alpha-demethylase is a cytochrome P450 enzyme of the cholesterol biosynthetic pathway belonging to the CYP51 gene family which is the most evolutionarily conserved member of the CYP superfamily. Mammalian (human, mouse, rat, pig) CYP51 genes are unique in sharing several common characteristics: highly conserved exon/intron borders and proximal promoter structures, ubiquitous expression at the highest level in the testis, and appearance of testis-specific transcripts that arise from differential polyadenylation site usage. CYP51 protein demethylates lanosterol to form follicular fluid meiosis-activating sterol, FF-MAS, which is, besides being an intermediate of cholesterol biosynthesis, also a signaling sterol that accumulates in ovaries. CYP51 protein resides in the endoplasmatic reticulum of most cells and also in acrosomal membranes of spermatids where transport through the Golgi apparatus is suggested. While sterol regulatory element binding protein (SREBP)-dependent transcriptional regulation of CYP51 contributes to synthesis of cholesterol, the germ-cell-specific cAMP/CREMtau-dependent upregulation might contribute to increased production of MAS.

Determination of reference genes for circadian studies in different tissues and mouse strains

BackgroundCircadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration.ResultsIn the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons.ConclusionsGenetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology.

Novel Insights into the Downstream Pathways and Targets Controlled by Transcription Factors CREM in the Testis

The essential role of the Crem gene in normal sperm development is widely accepted and is confirmed by azoospermia in male mice lacking the Crem gene. The exact number of genes affected by Crem absence is not known, however a large difference has been observed recently between the estimated number of differentially expressed genes found in Crem knock-out (KO) mice compared to the number of gene loci bound by CREM. We therefore re-examined global gene expression in male mice lacking the Crem gene using whole genome transcriptome analysis with Affymetrix microarrays and compared the lists of differentially expressed genes from Crem−/− mice to a dataset of genes where binding of CREM was determined by Chip-seq. We determined the global effect of CREM on spermatogenesis as well as distinguished between primary and secondary effects of the CREM absence. We demonstrated that the absence of Crem deregulates over 4700 genes in KO testis. Among them are 101 genes associated with spermatogenesis 41 of which are bound by CREM and are deregulated in Crem KO testis. Absence of several of these genes in mouse models has proven their importance for normal spermatogenesis and male fertility. Our study showed that the absence of Crem plays a more important role on different aspects of spermatogenesis as estimated previously, with its impact ranging from apoptosis induction to deregulation of major circadian clock genes, steroidogenesis and the cell-cell junction dynamics. Several new genes important for normal spermatogenesis and fertility are down-regulated in KO testis and are therefore possible novel targets of CREM.

CREM modulates the circadian expression of CYP51, HMGCR and cholesterogenesis in the liver

We show for the first time that isoforms of the cAMP response element modulator Crem, regulate the circadian expression of Cyp51 and other cholesterogenic genes in the mouse liver. In the wild type mice the expression of Cyp51, Hmgs, Fpps, and Sqs is minimal between CT12 and CT16 and peaks between CT20 and CT24. Cyp51, Fpps, and Sqs lost the circadian behavior in Crem-/- livers while Hmgcr is phase advanced from CT20 to CT12. This coincides with a phase advance of lathosterol/cholesterol ratio, as detected by GC-MS. Overexpression of CREMtau and ICER has little effect on the Hmgcr proximal promoter while they influence expression from the CYP51 promoter. Our data indicate that Crem-dependent regulation of Cyp51 in the liver results in circadian expression of this gene. We propose that cAMP signaling might generally be involved in the circadian regulation of cholesterol synthesis on the periphery.

Pre-cholesterol precursors in gametogenesis

Meiosis activating sterols (MAS) are biologically active post-lanosterol intermediates of cholesterol biosynthesis that are synthetized primarily in the gonads, including the sperm. MAS reinitiate the meiosis of oocytes in vitro while in vivo they seem to contribute to the oocyte quality and the progression of meiosis. The mRNAs for the MAS-producing enzyme lanosterol 14alpha-demethylase (CYP51) arise by alternative poly (A) signal selection. Only signals with low cleavage activity are used in the testis. Translation of mammalian CYP51s starts at one of the tandem in-frame ATGs. CYP51 protein of the bull is shorter compared to the human due to the usage of a more downstream translation start site. CYP51 proteins are post-translationally modified by glycosylations in the Golgi and on acrosomal membranes of the sperm. Green fluorescence protein-based ex vivo system has been developed to aid studying the intracellular transport of the MAS-producing CYP51. The influence of the post-translational modifications on MAS-synthesizing capacity is under investigation.