Thread by @GP_R101

If you are curious to find out how a holiday read was the 1st in a series of events ending up with the discovery of the first TADopathy in Endocrinology, one which explains the world's tallest giants, read this 🧵 (1/25)
tinyurl.com/ydb84laj

In between: 2 relocations, 3 kids born, a pandemic and a tragic loss...Let’s reconstruct the genesis of this long work!
It all started back in Jan 2017 during the Xmas holidays, when I first read an article published in the May 2015 issue of Cell tinyurl.com/y7l9rcl5👇(2/25)

It reported a new concept: the disruption of local chromatin domains called TADs and the resulting rewiring of gene-enhancers interaction is the pathomechanism behind some malformation syndromes. (3/25)

The concept was extremely intriguing considering what we already knew about the disease I study, X-linked acrogigantism (X-LAG): the huge expression levels of the GPR101 gene in the patients’ pituitary tumors.
tinyurl.com/y8sps2k7
(4/25)

However, we had no idea how the duplication of the genomic region harboring GPR101, associated with X-LAG, could lead to such high gene expression. We suspected it had to involve a cis-regulatory element such as a silencer or enhancer but didn’t know the mechanism. (5/25)

We also already knew from RNAseq studies that a gene fusion event was not the cause, so this newly reported pathomechanism seemed a plausible explanation. (6/25)

A few days later, back to work at the NIH, in Bethesda (MD, USA), I sent an email to the study’s PI, Dr Stefan Mundlos working at the MPIMG in Berlin (Germany). I announced my hypothesis and asked whether they were willing to collaborate. (7/25)

At the end of Jan, Dr Martin Franke, at the time a fellow in Mundlos lab, replied expressing enthusiasm for the requested collaboration and after examining the chromatin structure at the X-LAG locus agreed that my hypothesis was plausible! (8/25)

My supervisor, Dr Constantine Stratakis, was very supportive and we immediately set up a conf call, which was very instructive. (9/25)

That also prompted me to read Martin’s study published the previous year in Nature where the neo-TAD concept was 1st put forward
tinyurl.com/yawyf5u9 (10/25)

I started to culture patients’ cells and in Apr the first batch arrived in Berlin. Then, we had to wait until mid Nov for the first 4Cseq data to show that GPR101 displays indeed strong ectopic interactions with regions in neighboring centromeric TADs! (11/25)

This was a great proof of concept but based only on 1 patient. We needed to make sure it was a shared pathomechanism, not just a one-off event. (12/25)

So, I collected and cultured more patients and controls cells and grew them until there was enough for the 4C experiments. I also teamed up with my long-standing collaborator and buddy Dr Adrian Daly at the University of Liege. (13/25)

Together we were able to collect enough cells from 5 more patients (and controls). In Mar 2019, Martin, now working in the developmental and evolutionary genomics lab of Prof José Luis Gómez-Skarmeta in Seville (Spain), started to process the new samples. (14/25)

After hitting a few snafus, 1 year later we finally had the analyzed sequencing data. We now knew that neoTAD formation happens in multiple X-LAG patients! (15/25)

Then…well, we all know what happened worldwide 🦠
During that time, I managed to move back to Italy with my family just-in-time before lockdowns and started a new phase of my career at the Humanitas Research Hospital in Milan. (16/25)

Slowly but steadily the manuscript started to take its final form. Then, a tragic event occurred: the premature loss of Prof Gómez-Skarmeta 😔
tinyurl.com/y8v6vyub
Itching to report our findings and to honor his memory we were planning a submission for Nov 2020. (17/25)

But we knew that one piece of data was still missing: are there any enhancers at the X-LAG locus that regulate GPR101 expression when contacting its promoter within the neoTAD? And is the GPR101 promoter amenable to this regulation in relevant cell types? (18/25)

We felt that was the next logical step. So back to work! And with the help of a dedicated grad student, Eszter Trifan, we could answer those questions: YES, the GPR101 promoter permits the incorporation of new regulatory information (19/25)

and YES, one of the tested enhancers was active, at least in an embryonic context. We now reckoned we had a complete story and prepared for submission. We chose @AJHGNews for its outstanding quality and relevance to our study. (20/25)

@threadreaderapp unroll