To bind or not to bind – how to down-regulate target genes by liganded thyroid hormone receptor?

The terrain is well explored regarding genes whose gene expression is up-regulated upon binding of thyroid hormone (TH) to its nuclear receptor. This regulation mechanism has been intensively studied and is well understood. In contrast, a lot of white spots remain on the map when it comes to target genes whose expression is down-regulated upon binding of TH to the thyroid hormone receptor (TR). Since no consistent mechanism has been proposed to explain ligand-dependent down-regulation of target gene transcription several working hypotheses favour different molecular mechanisms. Some working theories suggest a direct binding of TR to regulatory elements of target genes. Others favour models that are independent of a direct DNA binding event. However recent data suggested that a direct binding of TR to DNA is dispensable for TH-dependent negative gene transcription

Functional cooperation between CREM and GCNF directs gene expression in haploid male germ cells

Cellular differentiation and development of germ cells critically depend on a coordinated activation and repression of specific genes. The underlying regulation mechanisms, however, still lack a lot of understanding. Here, we describe that both the testis-specific transcriptional activator CREMτ (cAMP response element modulator tau) and the repressor GCNF (germ cell nuclear factor) have an overlapping binding site which alone is sufficient to direct cell type-specific expression in vivo in a heterologous promoter context. Expression of the transgene driven by the CREM/GCNF site is detectable in spermatids, but not in any somatic tissue or at any other stages during germ cell differentiation. CREMτ acts as an activator of gene transcription whereas GCNF suppresses this activity. Both factors compete for binding to the same DNA response element. Effective binding of CREM and GCNF highly depends on composition and epigenetic modification of the binding site. We also discovered that CREM and GCNF bind to each other via their DNA binding domains, indicating a complex interaction between the two factors. There are several testis-specific target genes that are regulated by CREM and GCNF in a reciprocal manner, showing a similar activation pattern as during spermatogenesis. Our data indicate that a single common binding site for CREM and GCNF is sufficient to specifically direct gene transcription in a tissue-, cell type- and differentiation-specific manner

Intrauterine Growth Retarded Progeny of Pregnant Sows Fed High Protein:Low Carbohydrate Diet Is Related to Metabolic Energy Deficit

High and low protein diets fed to pregnant adolescent sows led to intrauterine growth retardation (IUGR). To explore underlying mechanisms, sow plasma metabolite and hormone concentrations were analyzed during different pregnancy stages and correlated with litter weight (LW) at birth, sow body weight and back fat thickness. Sows were fed diets with low (6.5%, LP), adequate (12.1%, AP), and high (30%, HP) protein levels, made isoenergetic by adjusted carbohydrate content. At −5, 24, 66, and 108 days post coitum (dpc) fasted blood was collected. At 92 dpc, diurnal metabolic profiles were determined. Fasted serum urea and plasma glucagon were higher due to the HP diet. High density lipoprotein cholesterol (HDLC), %HDLC and cortisol were reduced in HP compared with AP sows. Lowest concentrations were observed for serum urea and protein, plasma insulin-like growth factor-I, low density lipoprotein cholesterol, and progesterone in LP compared with AP and HP sows. Fasted plasma glucose, insulin and leptin concentrations were unchanged. Diurnal metabolic profiles showed lower glucose in HP sows whereas non-esterified fatty acids (NEFA) concentrations were higher in HP compared with AP and LP sows. In HP and LP sows, urea concentrations were 300% and 60% of AP sows, respectively. Plasma total cholesterol was higher in LP than in AP and HP sows. In AP sows, LW correlated positively with insulin and insulin/glucose and negatively with glucagon/insulin at 66 dpc, whereas in HP sows LW associated positively with NEFA. In conclusion, IUGR in sows fed high protein∶low carbohydrate diet was probably due to glucose and energy deficit whereas in sows with low protein∶high carbohydrate diet it was possibly a response to a deficit of indispensable amino acids which impaired lipoprotein metabolism and favored maternal lipid disposal

Nonstandard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty-nonstandard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for more reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Impaired Repressor Function in SUMOylation-Defective Thyroid Hormone Receptor Isoforms

BACKGROUND: Many nuclear receptors are modified by posttranslational modifications. OBJECTIVES: The transcriptional activity of thyroid hormone receptors (TRs) is modified by the influence of its ligand (thyroid hormones T3 and T4), but is also affected by posttranslational modifications. This study focuses on the SUMOylation of TR isoforms and the consequences on transcriptional activity and promoter occupancy. METHODS: SUMOylation of TR wild-type as well as isoform-specific point mutations have been studied in vitro. The promoter occupancy of TR (wild-type and double- or triple-mutated versions) and transcriptional cofactors have been investigated in chromatin immunoprecipitation (ChIP) and Re-ChIP analysis. RESULTS: TR is modified by SUMO proteins at defined residues: the isoform TRα is mainly modified at lysines 281 and 387, whereas lysines 50 and 443 are major SUMOylation sites of isoform TRβ. Lysine residues K281 (TRα) and K50 (TRβ) are isoform-specific SUMOylation sites influencing differing TR domains, whereas K387 (TRα) and K443 (TRβ) are orthologous residues. TRs are targets of all three SUMO variants (SUMO-1, -2, and -3). The transcriptional activity of SUMOylation-defective mutants of TR alters gene transcription from positively and negatively regulated T3 target genes. CONCLUSIONS: The most pronounced effect is an impaired repressor function of SUMOylation-deficient TR in the absence of T3. The transcriptional properties of SUMOylation-defective TRs can be at least in part ascribed to altered interaction with transcriptional cofactors such as SRC-1 and NCoR. Thus, these data indicate that posttranslational modification of TR by SUMOylation contribute to the fine tuning of its transcriptional response maintaining effects on cellular and physiological homeostasis

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis. The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV). For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red fluorescence, illustrating transfection success. Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques