Deletion mapping of regulatory elements for GATA3 in T cells reveals a distal enhancer involved in allergic diseases.

GATA3 is essential for T cell differentiation and is surrounded by genome-wide association study (GWAS) hits for immune traits. Interpretation of these GWAS hits is challenging because gene expression quantitative trait locus (eQTL) studies lack power to detect variants with small effects on gene expression in specific cell types and the genome region containing GATA3 contains dozens of potential regulatory sequences. To map regulatory sequences for GATA3, we performed a high-throughput tiling deletion screen of a 2 Mb genome region in Jurkat T cells. This revealed 23 candidate regulatory sequences, all but one of which is within the same topological-associating domain (TAD) as GATA3. We then performed a lower-throughput deletion screen to precisely map regulatory sequences in primary T helper 2 (Th2) cells. We tested 25 sequences with ∼100 bp deletions and validated five of the strongest hits with independent deletion experiments. Additionally, we fine-mapped GWAS hits for allergic diseases in a distal regulatory element, 1 Mb downstream of GATA3, and identified 14 candidate causal variants. Small deletions spanning the candidate variant rs725861 decreased GATA3 levels in Th2 cells, and luciferase reporter assays showed regulatory differences between its two alleles, suggesting a causal mechanism for this variant in allergic diseases. Our study demonstrates the power of integrating GWAS signals with deletion mapping and identifies critical regulatory sequences for GATA3.

Deletion of the homeodomain gene Six3 from kisspeptin neurons causes subfertility in female mice.

The homeodomain transcription factor SIX3 is a known regulator of eye, nose, and forebrain development, and has recently been implicated in female reproduction. Germline heterozygosity of SIX3 is sufficient to cause subfertility, but the cell populations that mediate this role are unknown. The neuropeptide kisspeptin is a critical component of the reproductive axis and plays roles in sexual maturation, ovulation, and the maintenance of gonadotropin secretion. We used Cre-Lox technology to remove Six3 specifically from kisspeptin neurons in mice to test the hypothesis that SIX3 in kisspeptin neurons is required for reproduction. We found that loss of Six3 in kisspeptin neurons causes subfertility and estrous cycle irregularities in females, but no effect in males. Overall, we find that SIX3 expression in kisspeptin neurons is an important contributor to female fertility.

Deletion of Six3 in post-proliferative neurons produces weakened SCN circadian output, improved metabolic function, and dwarfism in male mice.

OBJECTIVE: The increasing prevalence of obesity makes it important to increase the understanding of the maturation and function of the neuronal integrators and regulators of metabolic function. METHODS: Behavioral, molecular, and physiological analyses of transgenic mice with Sine oculis 3 (Six3) deleted in mature neurons using the Synapsincreallele. RESULTS: Conditional deletion of the homeodomain transcription factor Six3 in mature neurons causes dwarfism and weakens circadian wheel-running activity rhythms but increases general activity at night, and improves metabolic function, without impacting pubertal onset or fertility in males. The reduced growth in 6-week-old Six3fl/fl:Synapsincre (Six3syn) males correlates with increased somatostatin (SS) expression in the hypothalamus and reduced growth hormone (GH) in the pituitary. In contrast, 12-week-old Six3syn males have increased GH release, despite an increased number of the inhibitory SS neurons in the periventricular nucleus. GH is important in glucose metabolism, muscle function, and bone health. Interestingly, Six3syn males have improved glucose tolerance at 7, 12, and 18 weeks of age, which, in adulthood, is associated with increased % lean mass and increased metabolic rates. Further, 12-week-old Six3syn males have reduced bone mineralization and a lower bone mineral density, indicating that reduced GH levels during early life cause a long-term reduction in bone mineralization. CONCLUSION: Our study points to the novel role of Six3 in post-proliferative neurons to regulate metabolic function through SS neuron control of GH release.

Kiss1 is differentially regulated in male and female mice by the homeodomain transcription factor VAX1.

Regulation of Kiss1 transcription is crucial to the development and function of the reproductive axis. The homeodomain transcription factor, ventral anterior homeobox 1 (VAX1), has been implicated as a potential regulator of Kiss1 transcription. However, it is unknown whether VAX1 directly mediates transcription within kisspeptin neurons or works indirectly by acting upstream of kisspeptin neuron populations. This study tested the hypothesis that VAX1 within kisspeptin neurons regulates Kiss1 gene expression. We found that VAX1 acts as a repressor of Kiss1 in vitro and within the male arcuate nucleus in vivo. In female mice, we found that the loss of VAX1 caused a reduction in Kiss1 expression and Kiss1-containing neurons in the anteroventral periventricular nucleus at the time of the preovulatory luteinizing hormone surge, but was compensated by an increase in Kiss1-cFos colocalization. Despite changes in Kiss1 transcription, gonadotropin levels were unaffected and there were no impairments to fertility.