Savera J. Shetty

Sex Chromosome Complement and Gonadal Sex Influence Aggressive and Parental Behaviors in Mice

Across human cultures and mammalian species, sex differences can be found in the expression of aggression and parental nurturing behaviors: males are typically more aggressive and less parental than females. These sex differences are primarily attributed to steroid hormone differences during development and/or adulthood, especially the higher levels of androgens experienced by males, which are caused ultimately by the presence of the testis-determining gene Sry on the Y chromosome. The potential for sex differences arising from the different complements of sex-linked genes in male and female cells has received little research attention. To directly test the hypothesis that social behaviors are influenced by differences in sex chromosome complement other than Sry, we used a transgenic mouse model in which gonadal sex and sex chromosome complement are uncoupled. We find that latency to exhibit aggression and one form of parental behavior, pup retrieval, can be influenced by both gonadal sex and sex chromosome complement. For both behaviors, females but not males with XX sex chromosomes differ from XY. We also measured vasopressin immunoreactivity in the lateral septum, which was higher in gonadal males than females, but also differed according to sex chromosome complement. These results imply that a gene(s) on the sex chromosomes (other than Sry) affects sex differences in brain and behavior. Identifying the specific X and/or Y genes involved will increase our understanding of normal and abnormal aggression and parental behavior, including behavioral abnormalities associated with mental illness.

Gestational exposure to bisphenol a produces transgenerational changes in behaviors and gene expression.

Bisphenol A (BPA) is a plasticizer and an endocrine-disrupting chemical. It is present in a variety of products used daily including food containers, paper, and dental sealants and is now widely detected in human urine and blood. Exposure to BPA during development may affect brain organization and behavior, perhaps as a consequence of its actions as a steroid hormone agonist/antagonist and/or an epigenetic modifier. Here we show that BPA produces transgenerational alterations in genes and behavior. Female mice received phytoestrogen-free chow with or without BPA before mating and throughout gestation. Plasma levels of BPA in supplemented dams were in a range similar to those measured in humans. Juveniles in the first generation exposed to BPA in utero displayed fewer social interactions as compared with control mice, whereas in later generations (F(2) and F(4)), the effect of BPA was to increase these social interactions. Brains from embryos (embryonic d 18.5) exposed to BPA had lower gene transcript levels for several estrogen receptors, oxytocin, and vasopressin as compared with controls; decreased vasopressin mRNA persisted into the F(4) generation, at which time oxytocin was also reduced but only in males. Thus, exposure to a low dose of BPA, only during gestation, has immediate and long-lasting, transgenerational effects on mRNA in brain and social behaviors. Heritable effects of an endocrine-disrupting chemical have implications for complex neurological diseases and highlight the importance of considering gene-environment interactions in the etiology of complex disease.

Gestational Exposure to Low Dose Bisphenol A Alters Social Behavior in Juvenile Mice

Bisphenol A (BPA) is a man-made compound used to make polycarbonate plastics and epoxy resins; public health concerns have been fueled by findings that BPA exposure can reduce sex differences in brain and some behaviors. We asked if a low BPA dose, within the range measured in humans, ingested during pregnancy, would affect social behaviors in prepubertal mice. We noted sex differences in social interactions whereby females spent more time sitting side-by-side, while males engaged in more exploring and sitting alone. In addition BPA increased display of nose-to-nose contacts, play solicitations and approaches in both sexes. Interactions between sex and diet were found for self grooming, social interactions while sitting side-by-side and following the other mouse. In all these cases interactions were produced by differences between control and BPA females. We examined brains from embryos during late gestation to determine if gene expression differences might be correlated with some of the sexually dimorphic or BPA affected behaviors we observed. Because BPA treatments ended at birth we took the brains during embryogenesis to increase the probability of discovering BPA mediated effects. We also selected this embryonic age (E18.5) because it coincides with the onset of sexual differentiation of the brain. Interestingly, mRNA for the glutamate transporter, Slc1a1, was enhanced by exposure to BPA in female brains. Also we noted that BPA changed the expression of two of the three DNA methyltransferase genes, Dnmt1 and Dnmt3a. We propose that BPA affects DNA methylation of Sc1a1 during neural development. Sex differences in juvenile social interactions are affected by BPA and in particular this compound modifies behavior in females.

Roles of estrogen receptor α and androgen receptor in the regulation of neuronal nitric oxide synthase

In brain and peripheral tissues, steroid hormones regulate nitric oxide synthase (nNOS). We asked whether estrogen receptor‐α (ERα) and/or androgen receptor (AR) regulated nNOS immunoreactivity in mouse brain. First, we quantified cells singly labeled for nNOS immunoreactivity or labeled dually with ERα‐immunoreactive (‐ir) or AR‐ir cells in the nucleus accumbens (Acb), preoptic area (POA), bed nucleus of the stria terminalis (BNST), posterior dorsal and posterior ventral regions of the medial amygdala (MePD and MePV, respectively), and paraventricular nucleus (PVN). The POA and MePD contained the greatest number of double‐labeled cells. More nNOS‐ir cells were colabeled with ERα immunoreactivity compared with AR immunoreactivity. Next, by using a double mutant mouse in which males lacked functional ERα, AR, or both, we investigated the roles of these steroid receptors in nNOS‐ir cell numbers and immunoreactive area staining under testosterone (T) and estradiol (E2) conditions. Our data show that functional ERα is correlated with more nNOS‐ir cells under T conditions and more immunoreactive area staining in the POA under both T and E2 conditions. However, ERα decreases nNOS‐ir cell number in the BNST under E2 treatment. In summary, the data suggest that AR has organizational actions on nNOS‐ir cell numbers in the MePV, that interactions between ERα and AR genes occur in PVN, and that sex differences in nNOS‐ir area staining are limited to the POA. Thus, we show that ERα and AR interact to regulate nNOS in male and female brain in a site‐specific manner. J. Comp. Neurol. 453:336–344, 2002.

Estrogen Receptor β Regulates Sexually Dimorphic Neural Responses to Estradiol

Estrogen receptors (ERs) mediate many sexual dimorphisms in the neuroendocrine system and in behavior. We examined the consequences of the loss of functional estrogen receptor beta (ERbeta) on two sexually differentiated neural responses to estrogen. In wild type (WT) male mice, but not in females, estradiol (E(2)) treatment decreased estrogen receptor alpha immunoreactive (ERalpha-ir) cell numbers in the arcuate nucleus (ARC), the preoptic area (POA), and the ventromedial nucleus (VMN). These sex differences were reversed in ERbeta knockout (ERbetaKO) mice. Castrated ERbetaKOs did not show any change in ERalpha-ir cell number after E(2) treatment. Yet, E(2) decreased ERalpha-ir cell number in ovariectomized ERbetaKOs. Estradiol treatment increased progesterone receptor immunoreactive (PR-ir) cell number in WT female VMN and POA, but no change was noted in brains of WT castrates. In ERbetaKO mice the opposite relationship was found, E(2) treatment increased PR-ir cell number in male, but not in female, brains. Our results show that ERbeta influences several sexually dimorphic neural responses to estrogen. Moreover the data clearly show that ERbeta can modulate neural expression of ERalpha.

Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method

Transcription factor binding sites in chromatin are routinely inventoried by the chromatin immunoprecipitation assay, and these binding patterns can provide precise and detailed information about cell state. However, some fundamental molecular questions regarding transcription factor function require an understanding of in vivo binding dynamics as well as location information. Here we describe the crosslinking kinetics (CLK) assay, in which the time-dependence of formaldehyde crosslinking is used to extract on- and off-rates for chromatin binding in vivo.

The Modifier of Transcription 1 (Mot1) ATPase and Spt16 Histone Chaperone Co-regulate Transcription through Preinitiation Complex Assembly and Nucleosome Organization.

Modifier of transcription 1 (Mot1) is a conserved and essential Swi2/Snf2 ATPase that can remove TATA-binding protein (TBP) from DNA using ATP hydrolysis and in so doing exerts global effects on transcription. Spt16 is also essential and functions globally in transcriptional regulation as a component of the facilitates chromatin transcription (FACT) histone chaperone complex. Here we demonstrate that Mot1 and Spt16 regulate a largely overlapping set of genes in Saccharomyces cerevisiae. As expected, Mot1 was found to control TBP levels at co-regulated promoters. In contrast, Spt16 did not affect TBP recruitment. On a global scale, Spt16 was required for Mot1 promoter localization, and Mot1 also affected Spt16 localization to genes. Interestingly, we found that Mot1 has an unanticipated role in establishing or maintaining the occupancy and positioning of nucleosomes at the 5′ ends of genes. Spt16 has a broad role in regulating chromatin organization in gene bodies, including those nucleosomes affected by Mot1. These results suggest that the large scale overlap in Mot1 and Spt16 function arises from a combination of both their unique and shared functions in transcription complex assembly and chromatin structure regulation.

Crystal structure of the full Swi2/Snf2 remodeler Mot1 in the resting state

Swi2/Snf2 ATPases remodel protein:DNA complexes in all of the fundamental chromosome-associated processes. The single-subunit remodeler Mot1 dissociates TATA box-binding protein (TBP):DNA complexes and provides a simple model for obtaining structural insights into the action of Swi2/Snf2 ATPases. Previously we reported how the N-terminal domain of Mot1 binds TBP, NC2 and DNA, but the location of the C-terminal ATPase domain remained unclear (Butryn et al., 2015). Here, we report the crystal structure of the near full-length Mot1 from Chaetomium thermophilum. Our data show that Mot1 adopts a ring like structure with a catalytically inactive resting state of the ATPase. Biochemical analysis suggests that TBP binding switches Mot1 into an ATP hydrolysis-competent conformation. Combined with our previous results, these data significantly improve the structural model for the complete Mot1:TBP:DNA complex and suggest a general mechanism for Mot1 action.

An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking

Formaldehyde crosslinking is widely used in combination with chromatin immunoprecipitation (ChIP) to measure the locations along DNA and relative levels of transcription factor (TF)-DNA interactions in vivo. However, the measurements that are typically made do not provide unambiguous information about the dynamic properties of these interactions. We have developed a method to estimate binding kinetic parameters from time-dependent formaldehyde crosslinking data, called crosslinking kinetics (CLK) analysis. Cultures of yeast cells are crosslinked with formaldehyde for various periods of time, yielding the relative ChIP signal at particular loci. We fit the data using the mass-action CLK model to extract kinetic parameters of the TF-chromatin interaction, including the on- and off-rates and crosslinking rate. From the on- and off-rate we obtain the occupancy and residence time. The following protocol is the second iteration of this method, CLKv2, updated with improved crosslinking and quenching conditions, more information about crosslinking rates, and systematic procedures for modeling the observed kinetic regimes. CLKv2 analysis has been applied to investigate the binding behavior of the TATA-binding protein (TBP), and a selected subset of other TFs. The protocol was developed using yeast cells, but may be applicable to cells from other organisms as well.

Second-generation method for analysis of chromatin binding with formaldehyde–cross-linking kinetics

Formaldehyde-cross-linking underpins many of the most commonly used experimental approaches in the chromatin field, especially in capturing site-specific protein–DNA interactions. Extending such assays to assess the stability and binding kinetics of protein–DNA interactions is more challenging, requiring absolute measurements with a relatively high degree of physical precision. We previously described an experimental framework called the cross-linking kinetics (CLK) assay, which uses time-dependent formaldehyde–cross-linking data to extract kinetic parameters of chromatin binding. Many aspects of formaldehyde behavior in cells are unknown or undocumented, however, and could potentially affect CLK data analyses. Here, we report biochemical results that better define the properties of formaldehyde–cross-linking in budding yeast cells. These results have the potential to inform interpretations of “standard” chromatin assays, including chromatin immunoprecipitation. Moreover, the chemical complexity we uncovered resulted in the development of an improved method for measuring binding kinetics with the CLK approach. Optimum conditions included an increased formaldehyde concentration and more robust glycine-quench conditions. Notably, we observed that formaldehyde–cross-linking rates can vary dramatically for different protein–DNA interactions in vivo. Some interactions were cross-linked much faster than the in vivo macromolecular interactions, making them suitable for kinetic analysis. For other interactions, we found the cross-linking reaction occurred on the same time scale or slower than binding dynamics; for these interactions, it was sometimes possible to compute the in vivo equilibrium-binding constant but not binding on- and off-rates. This improved method yields more accurate in vivo binding kinetics estimates on the minute time scale.