DMRT1: An Ancient Sexual Regulator Required for Human Gonadogenesis

Abstract

Transcriptional regulators related to the invertebrate sexual regulators doublesex and mab-3 occur throughout metazoans and control sex in most animal groups. Seven of these DMRT genes are found in mammals, and mouse genetics has shown that one, Dmrt1, plays a crucial role in testis differentiation, both in germ cells and somatic cells. Deletions and, more recently, point mutations affecting human DMRT1 have demonstrated that its heterozygosity is associated with 46,XY complete gonadal dysgenesis. Most of our detailed knowledge of DMRT1 function in the testis, the focus of this review, derives from mouse studies, which have revealed that DMRT1 is essential for male somatic and germ cell differentiation and maintenance of male somatic cell fate after differentiation. Moreover, ectopic DMRT1 can reprogram differentiated female granulosa cells into male Sertoli-like cells. The ability of DMRT1 to control sexual cell fate likely derives from at least 3 properties. First, DMRT1 functionally collaborates with another key male sex regulator, SOX9, and possibly other proteins to maintain and reprogram sexual cell fate. Second, and related, DMRT1 appears to function as a pioneer transcription factor, binding "closed" inaccessible chromatin and promoting its opening to allow binding by other regulators including SOX9. Third, DMRT1 binds DNA by a highly unusual form of interaction and can bind with different stoichiometries.

Keywords: DM domain; DMRT1; DSD; Testis.

Figure 1:. The DM domain and DMRT1/DNA interaction

a. X-ray crystal structure of the human DMRT1 DM domain from Murphy et al. (2015) with major structural domains indicated. b. Conservation of metazoan DM domains. Positions of structural domains and sites of human mutations associated with DSD are indicated. c. X-ray crystal structure of human DMRT1 DM domains bound to DNA from Murphy et al. (2015), with DMRT1 protomers in distinct colors. Two protomers (pink and blue) insert in DNA major groove on one side of the binding site, while a single protomer (green) inserts in the major groove on the other side. Red oval indicates central basepair of the punctuated pseudopalindromic DNA site. d. DM domain amino acids affected by human DMRT1 point mutations associated with DSD. Affected amino acid side-chains are shown as space-filling models and labeled. R80 makes contact with the DNA phosphate backbone, Y84 is in the zinc module, and R111 makes contacts with conserved DNA bases in the recognition site.

Figure 2:. Sex-reversing R111G and R80S mutations reduce DNA binding by DMRT1 in vitro.

Left panel: DMRT1 protein translated in vitro was incubated with radiolabeled double-stranded DNAs previously shown to bind dimers, trimers or tetramers of DMRT1 as previously described [Murphy et al., 2015]. Reactions contained unprogrammed reticulocyte lysate (−), lysate programed with wild-type synthetic human Dmrt1 mRNA (WT), or synthetic mRNA encoding the R111G or R80S mutant DMRT1 proteins, as indicated. After incubation, reactions were subjected to native gene electrophoresis and autoradiography. Mobility of dimers, trimers and tetramers are indicated by arrowheads. The R111G mutation reduced trimer and dimer binding more severely than tetramer binding, while the R80S mutation reduced dimer and tetramer binding more severely than trimer binding. Right panel: DMRT1 proteins were incubated with DNA containing a DMRT1 binding site from the Foxl2 locus. Both mutations severely reduced binding to this site.

Figure 3:. Model of DMRT1 regulation of sexual cell fate maintenance and reprogramming.

Top: In sexual cell fate maintenance, DMRT1 (purple) occupies distal regulatory elements, in many cases with SOX9 (blue) and other regulators (brown). This binding helps keep chromatin accessible (“open”) and promotes expression of genes involved in Sertoli cell fate and function. Bottom: In reprogramming, DMRT1 binds inaccessible (“closed”) chromatin and promotes its conversion to a form accessible to SOX9 and other regulators. Occupancy by DMRT1 and other regulators relieves repression and promotes activation of genes in involved in Sertoli cell fate and function. How DMRT1 affects chromatin accessibility and potentially chromatin interactions is unknown, as are whether DMRT1 binding stoichiometry affects these functions and whether DMRT1 binding can promote formation of repressive chromatin at granulosa-expressed genes.