Close

Sex-determining 3D regulatory hubs revealed by genome spatial auto-correlation analysis

Irene Mota-Gómez, Juan Antonio Rodríguez, Shannon Dupont, Oscar Lao, Johanna Jedamzick, Ralf Kuhn, Scott Lacadie, Sara Alexandra García-Moreno, Alicia Hurtado, Rafael D. Acemel, Blanche Capel, Marc A. Marti-Renom, Darío G. Lupiáñez

Preprint posted on 18 November 2022 https://www.biorxiv.org/content/10.1101/2022.11.18.516861v1

Like Meta, but better. METALoci: a new tool to identify 3D regulatory regions in the genome, sex-determination edition.

Selected by Martin Estermann

Background:

During early embryonic development, the gonads of both male and female embryos develop similarly: they contain the same cell types and show highly similar gene expression. During a process known as gonadal sex determination, the bipotential gonad differentiates into two morphologically different organs: ovaries in XX females and testes in XY males. This process is regulated by two mutually exclusive genetic programs. The testicular program is initiated by the upregulation of the Y-linked gene Sry in XY supporting cells, which activates Sox9 expression. Sox9 then activates Fgf9, which is important in maintaining Sox9 expression, generating a positive feedback loop and promoting the differentiation towards Sertoli cells. In XX females, the lack of Sry results in the upregulation of Foxl2, initiating the differentiation towards the pre-granulosa fate and inhibiting Sox9 and Fgf9 expression.

Mutations on these key genes can result in differences of sex development (DSDs). DSDs include a wide range of conditions associated with a discordance between the genetic and phenotypical sex. The first step in the genetic diagnosis of DSDs involves the evaluation of a set of pre-selected mutations that are traditionally associated with DSDs. If unsuccessful, next-generation sequencing (NGS) technologies are used to identify novel mutations. Unfortunately, around 50% of DSDs patients won’t receive a definitive diagnosis. One of the biggest caveats of using these NGS technologies is the large amount of information that they generate, making it difficult to efficiently detect the mutation that can explain the pathogenesis. Additionally, whole-exome instead of whole-genome sequencing is typically performed, focusing only on the coding region of the genome, and excluding the non-coding or regulatory regions from the analysis. New technologies are needed to filter the large amount of data generated by NGS methods and associate the data with a potential phenotype.

In this preprint, the authors developed a methodology to identify 3D enhancer regions during gonadal cell differentiation by combining Hi-C and ChIP-seq data. Additionally, they were able to model in silico how different mutations affect gene expression and validate these results using animal models.

 

Key findings

1) METALoci: a new tool to identify 3D enhancer regions in the genome.

By integrating Hi-C and epigenetic data (H3K27Ac), the authors developed a new tool to identify regions in the genome that are physically close, as result of the 3D organization of the chromatin, and regulatory active. The authors used this methodology to identify changes during testicular differentiation, from supporting cell progenitors (E10.5) (Figure 1A) to Sertoli cells (E13.5) (Figure 1B). As can be seen, despite the 3D chromatin structure not changing much, there is a differential accumulation of epigenetic marks associated with open chromatin and active gene expression in the Sox9 locus after sex differentiation. This is consistent with previous data showing that Sox9 is a key driver of testicular differentiation. Additionally, the two main enhancers known to regulate Sox9 expression (Enh13 and TESCO) were also located in close proximity to Sox9 and displayed active chromatin marks. This data demonstrates the power of this tool to identify enhancer regions in the 3D space, correlated with gene expression.

Figure 1. METALoci changes during Sertoli cell sex differentiation. Graphical representation in 2D of the organization of the chromatin before (A) or after (B) sex differentiation. Here, a region of chromosome 11 is shown, based on the Hi-C data (left) and including the H3K27Ac data (right). This chromosomal region contains the Sertoli cell marker Sox9 and its reported enhancers TESCO and Enh13.

2) Identification of novel regulatory regions in gonadal differentiation.

Another important gene in testicular differentiation is Fgf9. Before this study, it was unknown how this gene is genetically regulated. Using METALoci, the authors identified a potential regulatory region. They computationally eliminated different 50kb regions from the locus and predicted how these mutations would potentially affect its 3D regulation and thus Fgf9 expression. This allowed the identification of a potential regulatory non-coding region located 250kb downstream of Fgf9. To validate these results, the authors generated a mouse model with a deletion in this genomic region (D306) and evaluated the effects of this deletion on gonadal sex differentiation. As can be seen in Figure 2, the deletion of this region resulted in the development of ovotestis or ovary-like structures in XY (male) individuals, expressing both testicular (SOX9) and ovarian (FOXL2) gonadal markers, at different degrees. This phenotype was the same as the one described for Fgf9-null mutants, confirming the importance of this regulatory region in controlling Fgf9 expression.

Figure 2. Gonadal differentiation in wild type and mutant embryos. Immunofluorescence for testicular (SOX9) and ovarian (FOXL2) supporting cell markers in E14.5 gonads from wild type male (XY, left), female (XX, right) and XY embryos with a deletion in Fgf9 enhancer region (middle).

Why I chose this paper:

As someone who studies gonadal differentiation and is interested in the diagnosis of DSDs, this paper resonated with me. I think that the technology the authors developed here can be used to close the gap between the genotype and the phenotype, one of the biggest issues in DSD diagnosis. I would love to see what other novel regulatory regions are detected using this tool. Additionally, the methodology seems to be straightforward enough to be applied not only to the gonadal development, but to other organs or even organisms. I can’t wait to see new applications for this tool.

 

Future directions / questions for the authors:

  • What are the minimum requirements to perform this kind of analysis? Could you combine Hi-C with ChIP-seq of other epigenetic marks, like H3K27me3, H3K9Ac or H3K4me?
  • Do you think that the METALoci tool can be applied, in the future, to genome sequencing data from DSDs patients to identify what mutations are responsible for changes in gene expression and thus the development of DSDs? What do you think would be necessary to achieve this goal?

 

 

Posted on: 13 December 2022

doi: https://doi.org/10.1242/prelights.33270

Read preprint (No Ratings Yet)

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the cell biology category:

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage

Also in the developmental biology category:

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

2nd Conference of the Visegrád Group Society for Developmental Biology

Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)

 



List by Nándor Lipták

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020

 



List by Jesus Victorino

Single Cell Biology 2020

A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.

 



List by Alex Eve

Society for Developmental Biology 79th Annual Meeting

Preprints at SDB 2020

 



List by Irepan Salvador-Martinez, Martin Estermann

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EDBC Alicante 2019

Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.

 



List by Sergio Menchero et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

SDB 78th Annual Meeting 2019

A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.

 



List by Alex Eve

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Young Embryologist Network Conference 2019

Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London

 



List by Alex Eve

Pattern formation during development

The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.

 



List by Alexa Sadier

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar
Close