Relationships between cortisol and urinary androgens in female titi monkeys (Plecturocebus cupreus).

Steroid hormones are critical to the regulation of sociosexual behavior. Their role in the formation of pair bonds is complicated by the relative scarcity of this social system in mammals, as well as species and taxonomic differences in endocrine systems. In the present study, we experimentally manipulated the hypothalamic-pituitary-adrenal axis in female titi monkeys (Plecturocebus cupreus), a neotropical monkey studied for its strong, selective pair bonds. We validated an assay for plasma and urinary cortisol in this species, showing a strong suppression of cortisol following dexamethasone injection, and a significant but somewhat blunted response to adrenocorticotrophin hormone (ACTH) stimulation. Urinary androgens did not change in response to dexamethasone or ACTH. Plasma and urinary cortisol were moderately correlated, whereas urinary cortisol and androgens were only correlated when extreme cortisol values were included. In this study, we laid groundwork for studying the role of glucocorticoids and androgens (and eventually, their interactions with peptides) in the behavioral endocrinology of pair bonds in female titi monkeys.

Histone H3.3 regulates dynamic chromatin states during spermatogenesis.

The histone variant H3.3 is involved in diverse biological processes, including development, transcriptional memory and transcriptional reprogramming, as well as diseases, including most notably malignant brain tumors. Recently, we developed a knockout mouse model for the H3f3b gene, one of two genes encoding H3.3. Here, we show that targeted disruption of H3f3b results in a number of phenotypic abnormalities, including a reduction in H3.3 histone levels, leading to male infertility, as well as abnormal sperm and testes morphology. Additionally, null germ cell populations at specific stages in spermatogenesis, in particular spermatocytes and spermatogonia, exhibited increased rates of apoptosis. Disruption of H3f3b also altered histone post-translational modifications and gene expression in the testes, with the most prominent changes occurring at genes involved in spermatogenesis. Finally, H3f3b null testes also exhibited abnormal germ cell chromatin reorganization and reduced protamine incorporation. Taken together, our studies indicate a major role for H3.3 in spermatogenesis through regulation of chromatin dynamics.

Anti-Müllerian hormone and inhibin-B concentrations vary cyclically in nonovulating queens within reference ranges established for determining gonadal status in cats.

OBJECTIVE: To define cyclic changes in anti-Müllerian hormone (AMH), inhibin-B, and progesterone concentrations and establish statistically valid, population-based clinical reference ranges in queens. ANIMALS: Cyclic queens (fertile, n = 6; infertile, 6) from an institutional breeding colony were blood sampled longitudinally, each for over 2 months, between November 2021 and February 2022, and residual serum samples from intact (n = 205) and ovariohysterectomized (49) queens from clinical submissions were used to establish reference ranges for intact and spayed females. METHODS: AMH and inhibin-B were measured using commercially available ELISAs, progesterone was measured using an in-house ELISA, and 90% CIs were calculated from these data. RESULTS: AMH and inhibin-B fluctuated in a highly correlated, cyclic pattern in 3 queens that did not ovulate immediately, whereas AMH declined as progesterone increased, indicative of ovulation, which occurred spontaneously early in the sampling period in 3 others; statistically valid reference ranges were established in intact and ovariohysterectomized females. CLINICAL RELEVANCE: Cyclic changes in hormone profiles were defined, providing relevant context for interpreting results in cases seeking to determine gonadal status (presence or absence of gonadal tissue) on the basis of established, population-based reference ranges reported here for cats for the first time.

myc maintains embryonic stem cell pluripotency and self-renewal.

While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditionally knocked out both c- and N-myc using myc doubly homozygously floxed mESC lines (cDKO). Both lines of myc cDKO mESC exhibited severely disrupted self-renewal, pluripotency, and survival along with enhanced differentiation. Chimeric embryos injected with DKO mESC most often completely failed to develop or in rare cases survived but with severe defects. The essential nature of myc for self-renewal and pluripotency is at least in part mediated through orchestrating pluripotency-related cell cycle and metabolic programs. This study demonstrates that endogenous myc genes are essential for mESC pluripotency and self-renewal as well as providing the first evidence that myc genes are required for early embryogenesis, suggesting potential mechanisms of myc contribution to iPS cell formation.

Chromatin immunoprecipitation assays for Myc and N-Myc.

Myc and N-Myc have widespread impacts on the chromatin state within cells, both in a gene-specific and genome-wide manner. Our laboratory uses functional genomic methods including chromatin immunoprecipitation (ChIP), ChIP-chip, and, more recently, ChIP-seq to analyze the binding and genomic location of Myc. In this chapter, we describe an effective ChIP protocol using specific validated antibodies to Myc and N-Myc. We discuss the application of this protocol to several types of stem and cancer cells, with a focus on aspects of sample preparation prior to library preparation that are critical for successful Myc ChIP assays. Key variables are discussed and include the starting quantity of cells or tissue, lysis and sonication conditions, the quantity and quality of antibody used, and the identification of reliable target genes for ChIP validation.

Endogenous mammalian histone H3.3 exhibits chromatin-related functions during development.

BACKGROUND: The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b. RESULTS: H3f3b knockout (KO) mice exhibit a semilethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in mouse embryonic fibroblasts (MEFs) with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile. CONCLUSIONS: Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development.

Myc and Miz-1 have coordinate genomic functions including targeting Hox genes in human embryonic stem cells.

BACKGROUND: A proposed role for Myc in maintaining mouse embryonic stem (ES) cell pluripotency is transcriptional repression of key differentiation-promoting genes, but detail of the mechanism has remained an important open topic. RESULTS: To test the hypothesis that the zinc finger protein Miz-1 plays a central role, in the present work we conducted chromatin immunoprecipitation/microarray (ChIP-chip) analysis of Myc and Miz-1 in human ES cells, finding homeobox (Hox) genes as the most significant functional class of Miz-1 direct targets. Miz-1 differentiation-associated target genes specifically lack acetylated lysine 9 and trimethylated lysine 4 of histone H3 (AcH3K9 and H3K4me3) 9 histone marks, consistent with a repressed transcriptional state. Almost 30% of Miz-1 targets are also bound by Myc and these cobound genes are mostly factors that promote differentiation including Hox genes. Knockdown of Myc increased expression of differentiation genes directly bound by Myc and Miz-1, while a subset of the same genes is downregulated by Miz-1 loss-of-function. Myc and Miz-1 proteins interact with each other and associate with several corepressor factors in ES cells, suggesting a mechanism of repression of differentiation genes. CONCLUSIONS: Taken together our data indicate that Miz-1 and Myc maintain human ES cell pluripotency by coordinately suppressing differentiation genes, particularly Hox genes. These data also support a new model of how Myc and Miz-1 function on chromatin.

N-Myc regulates expression of pluripotency genes in neuroblastoma including lif, klf2, klf4, and lin28b.

myc genes are best known for causing tumors when overexpressed, but recent studies suggest endogenous myc regulates pluripotency and self-renewal of stem cells. For example, N-myc is associated with a number of tumors including neuroblastoma, but also plays a central role in the function of normal neural stem and precursor cells (NSC). Both c- and N-myc also enhance the production of induced pluripotent stem cells (iPSC) and are linked to neural tumor stem cells. The mechanisms by which myc regulates normal and neoplastic stem-related functions remain largely open questions. Here from a global, unbiased search for N-Myc bound genes using ChIP-chip assays in neuroblastoma, we found lif as a putative N-Myc bound gene with a number of strong N-Myc binding peaks in the promoter region enriched for E-boxes. Amongst putative N-Myc target genes in expression microarray studies in neuroblastoma we also found lif and three additional important embryonic stem cell (ESC)-related factors that are linked to production of iPSC: klf2, klf4, and lin28b. To examine the regulation of these genes by N-Myc, we measured their expression using neuroblastoma cells that contain a Tet-regulatable N-myc transgene (TET21N) as well as NSC with a nestin-cre driven N-myc knockout. N-myc levels closely correlated with the expression of all of these genes in neuroblastoma and all but lif in NSC. Direct ChIP assays also indicate that N-Myc directly binds the lif promoter. N-Myc regulates trimethylation of lysine 4 of histone H3 in the promoter of lif and possibly in the promoters of several other stem-related genes. Together these findings indicate that N-Myc regulates overlapping stem-related gene expression programs in neuroblastoma and NSC, supporting a novel model by which amplification of the N-myc gene may drive formation of neuroblastoma. They also suggest mechanisms by which Myc proteins more generally contribute to maintenance of pluripotency and self-renewal of ESC as well as to iPSC formation.

N-Myc regulates a widespread euchromatic program in the human genome partially independent of its role as a classical transcription factor.

Myc proteins have long been modeled to operate strictly as classic gene-specific transcription factors; however, we find that N-Myc has a robust role in the human genome in regulating global cellular euchromatin, including that of intergenic regions. Strikingly, 90% to 95% of the total genomic euchromatic marks histone H3 acetylated at lysine 9 and methylated at lysine 4 is N-Myc-dependent. However, Myc regulation of transcription, even of genes it directly binds and at which it is required for the maintenance of active chromatin, is generally weak. Thus, Myc has a much more potent ability to regulate large domains of euchromatin than to influence the transcription of individual genes. Overall, Myc regulation of chromatin in the human genome includes both specific genes, but also expansive genomic domains that invoke functions independent of a classic transcription factor. These findings support a new dual model for Myc chromatin function with important implications for the role of Myc in cancer and stem cell biology, including that of induced pluripotent stem cells.

Methyl deficiency causes reduction of the methyl-CpG-binding protein, MeCP2, in rat liver.

MeCP2 is a member of a family of proteins [methyl- (cytosine-guanine)CpG-binding proteins] that bind specifically to methylated DNA and induce chromatin remodeling and gene silencing. Dietary deficiency of folate, choline and methionine causes decreased tissue S-adenosylmethionine concentrations (methyl deficiency), global DNA hypomethylation, hepatic steatosis, cirrhosis and ultimately hepatic tumorigenesis in rodents. We investigated the effects of this diet on expression of MeCP2 during pre-neoplastic transformation of liver tissue. After 9 weeks, MeCP2 mRNA level was slightly higher in methyl-deficient rats compared with replete controls, while after 36 weeks, a difference in MeCP2 mRNA level was no longer observed. In contrast, MeCP2 protein level was reduced almost 2-fold in the deficient rats compared with replete controls at both 9 and 36 weeks. Conversely, a second methyl-CpG-binding protein, MBD2, showed increased levels of both message and protein at the two time points. Low MeCP2 protein in the deficient rats was associated with a low level of the co-repressor protein, Sin3a, at 36 weeks. Moreover, a known gene target of MeCP2, the tumor suppressor gene metallothionein-I, was over-expressed in the deficient rat livers at both 9 and 36 weeks, suggesting that reduction in MeCP2 may have functional consequences. Methyl deficiency also caused an increase in the ratio of long to short variants of MeCP2 transcripts. This finding suggests that reduced MeCP2 protein level is the result of a reduced rate of translation. Reduction of MeCP2 protein expression may influence the initiation and/or progression of hepatic cancer induced by methyl deficiency and may provide a useful marker of pre-neoplastic change.