más aún para el comercio minorista
9. CONTACTOS DE INTERES
9.2 Organizaciones comerciales
The FK506-binding protein FKBP-12 is a cytosolic protein involved in protein-folding and chaperoning and is a pharmacological target for the immunosuppressant small-molecules FK506 and rapamycin 229,230. Another cellular protein, the mechanistic target of rapamycin
(mTOR), also known as FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1), binds to the FKBP-12-rapamycin complex with high affinity 231. This affinity is mediated by the FK506-rapamycin binding-domain (FRB) of mTOR/FRAP1. In a study using FKBP-12 fused to the GAL4 BD and FRB fused to GAL4 AD, researchers were able to induce expression of a reporter gene in a rapamycin-dependent manner in yeast 232. In a similar experiment in
mammalian cells, FRB was fused to the p65 activating domain, and FK506-12 was fused to the DNA binding domain of ZFHD1, a transcription factor from Arabidopsis that binds to a plant- specific DNA sequence 231,233. In both studies, dimerization of the fusion proteins and activation of the reporter gene only occurred in the presence of rapamycin. Because FKBP-12 also binds to the small-molecule FK506, addition of FK506 can antagonize the dimerization by competitive inhibition.
It was from these fundamental building blocks that Hathaway et al. created the Chromatin in vivo Assay (CiA) 234. The CiA was a tool developed to induce changes in the chromatin architecture of an endogenous gene in a chemically-dependent, reversible manner. The GAL4-UAS and ZFHD1- binding-sequence were inserted upstream of the Oct4 promoter in mouse embryonic stem cells, and the first exon of the Oct4 gene was replaced with a gene encoding nuclear EGFP. Using GAL4 BD fused to FKBP-12 and FRB fused to the
chromoshadow of mammalian HP1α (csHP1α), they succeeded in silencing gene expression of the Oct4-gfp reporter through the addition of rapamycin. This gene repression was accompanied by changes in histone posttranslational modifications that spanned kilobases from the GAL4- UAS. An increase in H3K9me3 and endogenous HP1γ at the locus was concurrent with a loss of the positive markers of gene expression, such as trimethylation of lysine 4 on histone H3
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quantitative polymerase chain reaction (qPCR). Reversal of the CIP and removal of the tethered csHP1α was achieved by removal of rapamycin and addition of FK506.
Using this system, Hathaway et al. observed how an induced heterochromatin domain behaved after removal of the ectopic stimulus of CIP and were able to develop a mathematical model for the maintenance of such a domain based on the propagation and loss of the H3K9me3 mark. Expansion of this assay into mouse embryo fibroblasts (MEFs) allowed for identification of cell-type specific factors that contributed to heterochromatin maintenance after multiple cell divisions, and the presence of two DNA binding arrays allowed for simultaneous recruitment of two different sets of fusion proteins to the locus 234. The CiA system has been used in subsequent studies to validate or screen for novel compounds that interrupt heterochromatin formation induced by HP1 proteins 235–237 and to screen for molecules that increase reactivation of Oct4 in differentiated cells 238.
Here, I report the construction of a modified Chromatin in vivo Assay that is modular and self-contained with interchangeable promoter, reporter gene, and CRISPR/Cas9-compatible homology arms. I used this next-generation CiA to characterize and compare the gene-silencing capacity of the human silencing hub (HUSH) complex subunit m-phase phosphoprotein 8 (MPP8) to the mammalian HP1 isoforms and to elucidate through comparison the importance of promoter structure on the maintenance of an induced heterochromatin domain. This assay will serve as yet another tool with which researchers can explore and contort the chromatin landscape in order to learn more about the suite of proteins that control the expression and repression of transcription through histone post-translational modifications, nucleosome positioning, or methylation of the underlying DNA.
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Table 1.1. Chromatin related proteins identified as suppressors of variegation. Data obtained from Flybase.org, FB2019_03, released Jun 11, 2019
Symbol UniProt Function H. sapiens orthologs M. musculus orthologs S. cerevisiae orthologs
AGO2 Essential for RNA interference (RNAi). AGO3 , AGO1 , AGO4 , PIWIL2 , AGO2 , PIWIL3 , PIWIL1 , PIWIL4
Piwil2 , Ago4 , Piwil4 , Ago2 ,
Piwil1 , Ago3 , Ago1 CUR1 , BTN2
ash1 Trithorax group (TrxG) protein
SETD2 , SETBP1 , DIDO1 , NSD2 , NSD1 , SETD1A , SETD5 , KMT2C , PHF20L1 , KMT2D , ASH1L , KMT2E , NSD3 , SETD1B , PHF20
Setd5 , Spocd1 , Kmt2e , Phf20l1 , Setbp1 , Setd1b , Nsd1 , Phf3 , Kmt2c , Nsd3 , Setd1a , Ash1l , Nsd2 , Setd2 , Dido1 , Kmt2d
CTI6 , BYE1 , SET3 , SET4 , SET1 , RSC2 , SET2
aub Acts via the piwi-interacting RNA (piRNA) metabolic process
AGO4 , PIWIL2 , AGO2 , PIWIL3 , PIWIL1 , PIWIL4 , AGO1 , AGO3
Piwil4 , Ago2 , Ago4 , Piwil2 ,
Piwil1 , Ago1 -
Dcr-1 Essential for RNA interference (RNAi). DICER1 , FANCM , DHX58 Dhx58 , Fancm , Dicer1 MPH1 , IRC3
E(z) Polycomb group (PcG) protein SETD7 , EZH2 , EZH1 Ezh2 , Setd7 , Ezh1 SET2
egg Histone methyltransferase that SETDB2 , SETDB1 Setdb1 , Setdb2 SET2
gpp Histone methyltransferase. DOT1L Dot1l DOT1
HDAC1
Catalyzes the deacetylation of lysine residues on the N- terminal part of the core histones (H2A, H2B, H3 and H4).
HDAC3 , HDAC1 , HDAC2 ,
HDAC8 Hdac2 , Hdac1 , Hdac3 , Hdac8 RPD3 , HOS1 , HOS2
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Kdm4A Probable histone demethylase KDM4D , KDM4A , KDM4E , KDM4C , KDM4B Kdm4a , Kdm4c , Kdm4b , Kdm4d GIS1 , RPH1 Kdm4B Probable histone demethylase KDM4C , KDM4D , KDM4E , KDM4B , KDM4A Kdm4a , Kdm4d , Kdm4b , Kdm4c GIS1 , RPH1 MRG15 Part of the Tip60 chromatin-remodeling
complex.
MORF4L2 , ARID4A , ARID4B , MSL3 , MORF4L1
Msl3l2 , Gm5073 , Arid4a , Morf4l2 , Morf4l1 , Arid4b ,
Msl3 , Morf4l1b EAF3
Pc Polycomb group (PcG) protein. CBX2 , CBX6 , CBX8 , CBX7 , CBX4 Cbx7 , Npcd , Cbx2 , Cbx4 , Cbx6 , Cbx8 -
PCNA Auxiliary protein of DNA polymerase delta PCNA Pcna POL30
ph-p Polycomb group (PcG) protein. SAMD11 , PHC1 , SAMD13 , SAMD7 , PHC3 , SCML2 , PHC2 , SAMD1 , M6PR
Phc1 , Samd11 , Phc3 , Samd1 ,
Phc2 , Samd7 -
PR-Set7 Histone methyltransferase KMT5A Kmt5a -
Sce E3 ubiquitin-protein ligase. PCGF3 , RING1 , RNF2 , BRCA1 Brca1 , Pcgf3 , Traip , Ring1 , Rnf2 -
Sirt1 NAD-dependent histone deacetylase SIRT1 , SIRT3 , SIRT5 Sirt3 , Sirt5 , Sirt2 , Sirt1 HST4 , SIR2 , HST3 , HST2 , HST1
Su(var)205 Structural component of heterochromatin
CDY1 , CBX5 , CDY2A , CDY1B , CDYL , CBX3 , CDY2B , CDYL2 ,
MPHOSPH8 , CBX1 , CBX7
Cbx7 , Cbx5 , Cdyl2 , Cbx3 ,
Mphosph8 , Cdyl , Cbx1 NEW1
Su(var)3-3 Probable histone demethylase SMOX , MAOB , KDM1B , PAOX , KDM1A , MAOA Maob , Maoa , Kdm1b , Paox , Kdm1a , Smox FMS1 Su(var)3-9 Histone methyltransferase PRSS48 , SUV39H1 , EHMT1 , SETMAR ,
EHMT2 , SUV39H2
Suv39h1 , Eif2s3x , Tpsb2 , Setmar , Suv39h2 , Ehmt2 ,
Ehmt1 GCD11 , SET2
Su(z)12 Polycomb group (PcG) protein.
SUZ12
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trx Histone methyltransferase PHF11 , KMT2A , KMT2B , PHF7 Phf11a , Phf11c , Gm6904 , Kmt2a , Phf11b , Phf11d ,
Kmt2b , Phf7 SET1
XNP Modifies gene expression by affecting chromatin (Potential).
DNMT3B , RAD54L2 ,
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