Lab Notes

Back to Andy Ultimate goal: Identify genes that are trisomy-related but differentially expressed based on parent-of-origin. 1) Identify trisomic genes 2)

Methods followed for various experiments: Workflow for candidate gene identification: Start with P_PT and M_MT gene lists -> find all overlapping genes (these should represent all trisomic genes plus some noise) -> Identify genes that are up-regulated in one parent and down-regulated in another

Identified the overlapping genes with the top and bottom 10% logFC values and we are especially interested in genes that have an inverse correlation for M v P.

Fold change is First/Second, so a -FC for a PT/MT comparison means the PT is less than the MT

Some Ensembl IDs map to the same gene but represent alternative splicing events (http://vega.sanger.ac.uk/Mus_musculus/Gene/Summary?db=core;g=OTTMUSG00000028340;r=16:91647506-91679221)

Dimentia/AD in DS- "there is a subset of aged DS persons who do not appear to develop clinical signs of dementia at any age." AD in Down

Excel: Gene names need to be truncated using command- left(A1, 18)


Methods for Fus Investigation Started with paper http://illiad.davidson.edu/pdf/283460.pdf FUS Overview
-> They describe many mutations of the FUS protein and how they can affect neuronal cells. Needed to verify that at least some of the mutations were producing inept proteins (so the data can be applied to transcription levels of healthy protein)
->this was accomplished by analyzing all reported mutations in their supplementary data table of mutation and cross-referencing them with GenePeeks research tool Fus Results to find uncontested pathologic nonsense mutations.
Tremors in ClinVar Our mouse model shows tremors and a nonsense mustation is implicated in these sorts of tremors (could be from down regulation?)

Many of the investigated genes,Wdr6,Ubtf,Luc7l3,Fus, are implicated in broad transcriptional functions. Andy Genes:
Up/Down Genes Identified:

• Ccng1 Cyclin gene, involved in the cell cycle and unlikely to be of interest here
• Hnrnpk Found with FUS string
• Zbtb26 Not enough Info
• Wdr6 WD repeats are minimally conserved regions of approximately 40 amino acids typically bracketed by gly-his and trp-asp (GH-WD), which may facilitate formation of heterotrimeric or multiprotein complexes. The encoded protein interacts with serine/threonine kinase 11, and is implicated in cell growth arrest.
  
• Zxdc Not enough information
• Gm12790 predicted gene- not enough info
• Ubtf - The encoded protein plays a critical role in ribosomal RNA transcription as a key component of the pre-initiation complex, mediating the recruitment of RNA polymerase I to rDNA promoter regions. The encoded protein may also play important roles in chromatin remodeling and pre-rRNA processing, and its activity is regulated by both phosphorylation and acetylation.
  
• Luc7l3 This protein localizes with a speckled pattern in the nucleus, and could be involved in the formation of splicesome via the RE and RS domains.
• Gm11800 Predicted gene- not enough info
• Cox17 cytochrome oxidase (COX) is a key complex found in the mitchondrial respiration chain. Cox17 is an assembly protein that helps this complex form and is thought to recruit copper to the complex, which helps activate it. The protein is ubiquitously produced in cells and implicated in embryonic development. Book; pg 134) According to NCBI and gene, this gene is no longer a candidate for COX deficiency

• Gm14300 predicted gene- not enough info
• FusThis gene encodes a multifunctional protein component of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. The hnRNP complex is involved in pre-mRNA splicing and the export of fully processed mRNA to the cytoplasm. This protein belongs to the FET family of RNA-binding proteins which have been implicated in cellular processes that include regulation of gene expression, maintenance of genomic integrity and mRNA/microRNA processing.(GENE) Defects in this gene are implicated in ALS(6) ASL causes
  
• Gm14296 predicted gene- not enough info
• Nop56 a Protein Coding gene. Diseases associated with NOP56 include Spinocerebellar Ataxia 36 and Spinocerebellar Ataxia Type36. Among its related pathways are Chaperonin-mediated protein folding and rRNA processing in the nucleus and cytosol. GO annotations related to this gene include poly(A) RNA binding and snoRNA binding.
• Hnrnpr THIS ONE INTERACTS WITH FUS String

• Rsrp1 arginine and serine rich protein 1
• Rhox5

Same Direction (Up/up or down/down) and in P_M:

• Gabpa - This gene encodes one of three GA-binding protein transcription factor subunits which functions as a DNA-binding subunit. Because of its chromosomal localization and ability to form heterodimers with other polypeptides, this gene may play a role in the Down Syndrome phenotype.
  
• Dyrk1a - Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family; localized in the Down syndrome critical region of chromosome 21, and is considered to be a strong candidate gene for learning defects associated with Down syndrome; It catalyzes its autophosphorylation on serine/threonine and tyrosine residues. It may play a significant role in a signaling pathway regulating cell proliferation and may be involved in brain development.
  
• Cryzl1
  
• Scaf8 (HSA6)
  
• Jam2
  

Jam2 and Gabpa are proximal to one another:

Same Direction (Up/up or down/down) and not in P_M:

• Ets2 - HSA 21 - This gene encodes a transcription factor which regulates genes involved in development and apoptosis. The encoded protein is also a protooncogene and shown to be involved in regulation of telomerase.
  
• Dscr3 - HSA21 - The DSCR3 (Down syndrome critical region gene 3) gene is found in this region and is predictated to contain eight exons. DSCR3 is expressed in most tissues examined. (this link also characterized the critical region).
  
• Chaf1b- HSA21 - Chromatin assembly factor I (CAF-I) is required for the assembly of histone octamers onto newly-replicated DNA. CAF-I is composed of three protein subunits, p50, p60, and p150. The protein encoded by this gene corresponds to the p60 subunit and is required for chromatin assembly after replication. The encoded protein is differentially phosphorylated in a cell cycle-dependent manner. In addition, it is normally found in the nucleus except during mitosis, when it is released into the cytoplasm. This protein is a member of the WD-repeat HIR1 family and may also be involved in DNA repai.
  
• Rcan1 - HSA21 - The protein encoded by this gene interacts with calcineurin A and inhibits calcineurin-dependent signaling pathways, possibly affecting central nervous system development. This gene is located in the minimal candidate region for the Down syndrome phenotype, and is overexpressed in the brain of Down syndrome fetuses. Chronic overexpression of this gene may lead to neurofibrillary tangles such as those associated with Alzheimer disease.-- Chronic overexpression is suspected to play a role in alzheimers.
  
• Atp5o- HSA21 - The protein encoded by this gene is a component of the F-type ATPase found in the mitochondrial matrix. F-type ATPases are composed of a catalytic core and a membrane proton channel. The encoded protein appears to be part of the connector linking these two components and may be involved in transmission of conformational changes or proton conductance.
  
• Paxbp1 - HSA21 - This gene encodes a protein that may bind to GC-rich DNA sequences, which suggests its involvement in the regulation of transcription. Alternative splicing of this gene results in multiple transcript variants.
  
• Wrb - HSA21 - Since the region proposed to contain the gene(s) for congenital heart disease (CHD) in Down syndrome (DS) patients has been restricted to 21q22.2-22.3, this gene, which maps to 21q22.3, has a potential role in the pathogenesis of Down syndrome congenital heart disease.--- Paper from class found no Parent-of-origin transcriptional effects with WRB (tho methylation patterns varied). *This study should be re-examined to determine if the WRB
• Rps6ka2
• Fgfr1op
• Bach1
• Cct8
• Fcgr2b
• Ice2
• Tdgf1
• Rwdd2b
• Ltn1
• Srrt - HSA7 - Acts as a mediator between the cap-binding complex (CBC) and the primary microRNAs (miRNAs) processing machinery during cell proliferation. Contributes to the stability and delivery of capped primary miRNA transcripts to the primary miRNA processing complex containing DGCR8 and DROSHA, thereby playing a role in RNA-mediated gene silencing (RNAi) by miRNAs.(GeneCards).
• Hmgn1
• Ezr
• Tmem181a
• Mis18a
• Gtf2h5
• Brwd1 - HSA21 - This gene encodes a member of the WD repeat protein family. WD repeats are minimally conserved regions of approximately 40 amino acids typically bracketed by gly-his and trp-asp (GH-WD) residues which may facilitate formation of heterotrimeric or multiprotein complexes. Members of this family are involved in a variety of cellular processes including cell cycle progression, signal transduction, apoptosis, and gene regulation. This protein contains 2 bromodomains and multiple WD repeats. This gene is located within the Down syndrome region-2 on chromosome 21.
• Son - HSA21 - This gene encodes a protein that contains multiple simple repeats. The encoded protein binds RNA and promotes pre-mRNA splicing, particularly of transcripts with poor splice sites. The protein also recognizes a specific DNA sequence found in the human hepatitis B virus (HBV) and represses HBV core promoter activity.
  
• Mrpl39 - HSA21 - Mitochondrial ribosome - his gene encodes a 39S subunit protein. Two transcript variants encoding distinct isoforms have been described.
  
• Ttc3 - HSA21 - E3 ubiquitin-protein ligase that mediates the ubiquitination and subsequent degradation of phosphorylated Akt (AKT1, AKT2 and AKT3) in the nucleus. Acts as a terminal regulator of Akt signaling after activation; its phosphorylation by Akt, which is a prerequisite for ubiquitin ligase activity, suggests the existence of a regulation mechanism required to control Akt levels after activation. Catalyzes the formation of 'Lys-48'-polyubiquitin chains. May play a role in neuronal differentiation inhibition via its interaction with CIT. (UniProt)
  
• Prdx2
• Hk1 - HSA10 -
• Urb1 - HSA21 -
• Psmg1 - HSA21 -
• Ggnbp2 - HSA17 - Minimal information
  
• Gart - HSA21 - The protein encoded by this gene is a trifunctional polypeptide. It has phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase activity which is required for de novo purine biosynthesis. This enzyme is highly conserved in vertebrates.
  
• Atxn1
• Dynlt1b - HSA6 - This gene encodes a component of the motor complex, cytoplasmic dynein, which transports cellular cargo along microtubules in the cell. The encoded protein regulates the length of primary cilia which are sensory organelles found on the surface of cells.

Ben Genes:

• RIPK4- mutations cause severe phenotype (wrinkly skin, fused eyelids, small mouth, no nose) and babies do not survive. The gene is implicated in keratinocyte differentiation. RIPK4 directly regulates IRF6-> differentiation in keratinocytes. (Not very promising for trisomy)

• Usp16- triplication of Usp16 reduces the self-renewal of haematopoietic stem cells and the expansion of mammary epithelial cells, neural progenitors and fibroblasts.Usp16 can remove ubiquitin from histone H2A on lysine 119, a critical mark for the maintenance of multiple somatic tissues. Downregulation of Usp16, either by mutation of a single normal Usp16 allele or by short interfering RNAs, largely rescues all of these defects.Furthermore, in human tissues overexpression of USP16 reduces the expansion of normal fibroblasts and postnatal neural progenitors, whereas downregulation of USP16 partially rescues the proliferation defects of Down's syndrome fibroblasts. Taken together, these results suggest that USP16 has an important role in antagonizing the self-renewal and/or senescence pathways in Down's syndrome and could serve as an attractive target to ameliorate some of the associated pathologies.

1. 1. 1. 1. All gene info form NIH Gene database except when specified