The GAGA factor regulatory network: Identification of GAGA factor associated proteins

PLOS ONE, Dec 2019

The Drosophila GAGA factor (GAF) has an extraordinarily diverse set of functions that include the activation and silencing of gene expression, nucleosome organization and remodeling, higher order chromosome architecture and mitosis. One hypothesis that could account for these diverse activities is that GAF is able to interact with partners that have specific and dedicated functions. To test this possibility we used affinity purification coupled with high throughput mass spectrometry to identify GAF associated partners. Consistent with this hypothesis the GAF interacting network includes a large collection of factors and complexes that have been implicated in many different aspects of gene activity, chromosome structure and function. Moreover, we show that GAF interactions with a small subset of partners is direct; however for many others the interactions could be indirect, and depend upon intermediates that serve to diversify the functional capabilities of the GAF protein.

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The GAGA factor regulatory network: Identification of GAGA factor associated proteins

March The GAGA factor regulatory network: Identification of GAGA factor associated proteins Dmitry Lomaev 1 2 Anna Mikhailova 1 2 Maksim Erokhin 1 2 Alexander V. Shaposhnikov 1 2 James J. Moresco 2 Tatiana Blokhina 1 2 Daniel Wolle 0 2 Tsutomu Aoki 0 2 Vladimir Ryabykh 2 John R. Yates 2 Yulii V. Shidlovskii 1 2 Pavel Georgiev 1 2 Paul Schedl 0 1 2 Darya Chetverina 1 2 0 Department of Molecular Biology Princeton University , Princeton, NJ , United States of America, 4 Institute of Animal Physiology , Biochemistry and Nutrition, Borovsk , Russia 1 Institute of Gene Biology, Russian Academy of Sciences , Moscow , Russia , 2 Department of Chemical Physiology, SR302B, The Scripps Research Institute , La Jolla, California , United States of America 2 Editor: Giacomo Cavalli, Centre National de la Recherche Scientifique , FRANCE The Drosophila GAGA factor (GAF) has an extraordinarily diverse set of functions that include the activation and silencing of gene expression, nucleosome organization and remodeling, higher order chromosome architecture and mitosis. One hypothesis that could account for these diverse activities is that GAF is able to interact with partners that have specific and dedicated functions. To test this possibility we used affinity purification coupled with high throughput mass spectrometry to identify GAF associated partners. Consistent with this hypothesis the GAF interacting network includes a large collection of factors and complexes that have been implicated in many different aspects of gene activity, chromosome structure and function. Moreover, we show that GAF interactions with a small subset of partners is direct; however for many others the interactions could be indirect, and depend upon intermediates that serve to diversify the functional capabilities of the GAF protein. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction The Drosophila GAGA factor (GAF) is an unusually versatile DNA binding protein that functions in remarkably diverse range of regulatory contexts. GAF was first identified as a transcriptional activator in in vitro transcription experiments with the Ultrabithorax (Ubx) and engrailed (en) genes. It bound to GAGAG motifs in the promoter region and stimulated transcription [1±3]. Consistent with a function in transcriptional activation, mutations in the gene encoding GAF, Trithorax-like (Trl), were shown to dominantly enhance the haploinsufficiency of the Ubx gene [ 4 ]. Moreover, the Trl mutations also dominantly enhanced position effect variegation (PEV) [ 4 ]. While these findings suggested that GAF functions as a conventional transcriptional activator, in vitro chromatin assembly experiments pointed to a rather different and unexpected role. When GAF was included in chromatin assembly assays using a plasmid containing the hsp70 gene as the DNA template, it was found to mediate the formation of a nucleosome free region spanning the GAF binding motifs in the hsp70 promoter [ 5 ]. The GAF factor helped recruit chromatin remodeling complexes to the template, and then functioned to exclude nucleosomes from the exposed promoter sequence [ 6 ]. Amongst the remodeling complexes that are thought to function together with GAF are PBAP, NURF and FACT [7±10]. A role in the formation/maintenance of nucleosome free regions of chromatin in vitro is recapitulated in vivo in transgene experiments with the hsp26 and hsp70 genes [ 11,12 ]. In addition to ensuring that promoter sequences are accessible, GAF is thought to play a more direct role in transcription by regulating promoter pausing [13±15]. These are not, however, the only known biological activities of the GAF protein. It has also been implicated in Polycomb group (PcG) dependent silencing [16±18], chromosome condensation and segregation during mitosis [19] and boundary activity [ 20 ]. Consistent with these multiple functions, GAF binding sequences are found in promoters, enhancers, Polycomb response elements (PREs) and boundary elements, while chromatin immunoprecipitation experiments localize GAF protein to these elements in vivo [21±26]. It is not yet understood how GAF carries out this diverse array of functions. The GAF protein itself has a relatively simple structure. It has an N-terminal BTB/POZ domain, a central C2H2-type zinc finger and several alternative glutamine rich (Q) C-terminal domains. The single zinc finger domain is responsible for DNA binding to the GAGAG pentanucleotide [ 27 ]. As there is little apparent flexibility in the DNA recognition properties of GAF, a plausible idea is that its different activities depend upon the ability of the GAF protein to interact either directly or indirectly with multiple partners. There is already evidence supporting this possibility. The GAF BTB/POZ domain has been shown to mediate protein-protein interactions and it participates in the formation of homo-oligomers and hetero-oligo (...truncated)


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Dmitry Lomaev, Anna Mikhailova, Maksim Erokhin, Alexander V. Shaposhnikov, James J. Moresco, Tatiana Blokhina, Daniel Wolle, Tsutomu Aoki, Vladimir Ryabykh, John R. Yates, Yulii V. Shidlovskii, Pavel Georgiev, Paul Schedl, Darya Chetverina. The GAGA factor regulatory network: Identification of GAGA factor associated proteins, PLOS ONE, 2017, Volume 12, Issue 3, DOI: 10.1371/journal.pone.0173602