[http://gcat.davidson.edu/mediawiki-1.27.1/index.php/Andy 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." [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184282/ AD in Down]

Excel: Gene names need to be truncated using command- left(A1, 18) 
Andy Genes: 
Up/Down Genes Identified: 
* Ccng1
* Hnrnpk
* Zbtb26
* Wdr6
* Zxdc
* Gm12790
* Ubtf
* Luc7l3
* Gm11800
* Cox17
* Gm14300
* Fus
* Gm14296
* Nop56
* Hnrnpr
* Rsrp1
* 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 
<center>
Jam2 and Gabpa are proximal to one another: 
[[File:Jam2gapba.jpg | 700px]]

</center>
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.

####All gene info form NIH Gene database except when specified

Lab Notes

2017-02-28T15:29:48Z

Anbaay:

Lab Notes

2017-02-28T03:44:35Z

Anbaay:

Lab Notes

2017-02-27T04:18:08Z

Anbaay:

[Back to Andy, http://gcat.davidson.edu/mediawiki-1.27.1/index.php/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

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

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.

2017-02-21T15:59:59Z

Anbaay:

Lab Notes

2017-02-16T15:34:51Z

Anbaay:

Lab Notes

2017-02-14T15:55:08Z

Anbaay:

Lab Notes

2017-02-07T15:11:58Z

Anbaay:

Methods followed for various experiments:

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

2017-02-07T14:58:39Z

Anbaay:

[[Lab Notes]]

Assignment for Tuesday: Run a trisomic vs disomic comparison.

Interpreting Data:
What do we want to look at? How do we make sense of the numbers? Fold change is important but so is base mean transcription level.
 The first data set entered will be the numerator of any fold change numbers!

<center>
It's being created right now: 
[[File:ScreenGrab.png]]
</center>

Write introduction that conveys understanding of the study we will be conducting.
Looking at mice that have been controlled for maternal vs paternal trisomy inheritance. We have the transcription data and are looking for differential expression

Good trisomy info with some genes of interest:
http://www.ds-health.com/trisomy.htm

stylizing: human genes are all caps and italicized
non-human are only italicized

proteins are the same name but not italicized

 PAPER NOTES 

“Trisomy 21 Alters DNA Methylation in Parent- of-Origin-Dependent and -Independent Manners”
In this article, researchers found a reliable way to distinguish maternal and paternal inheritance of the extra chromosome that produces Down syndrome. This method relies on the gene imprinting in a differentially methylated region named WRB, and specifically in CpG island 2. Because we are working with embryonic stem cells we expect this region to hypomethylated and uninformative as to the
Researchers reported a parent-of-origin-dependent methylation status at two genes, RUNX1 and TMEM131. RUNX1 was found to be differentially methylated independent of parent of origin when compared to disomic individuals. This means it could function as a marker by which to check the
WRB on was found to be differentially methylated based on parent of origin, but only in mature cells and not in embryonic stem cells. This makes the promise of finding differential expression in our mouse embryonic stem cells unlikely, but we should investigate anyway, to find out if this non-differntial methylation is conserved in our embryonic stem cells. Transcriptome analysis of WRB in embryonic stem cells revealed biallelic expression and so this should be investigated in our experiments as well.

“Domains of genome-wide gene expression dysregulation in Down’s syndrome”
Some down syndrome models are based only on the triplication of certain genes (SIM2, DYRK1A). The main differences of expression between discordant twins were clustered in differential gene expression domains. EdgeR used to evaluate the differential expression between the twins’ fibroblast cells. When comparing the T1DS to T2N, 337 GEDDs (Gene expression disregulation domains).
When creating iPS cells from the twin fibroblasts, researchers found that the GEDD differential expression was largerly preserved and concluded that “GEDDs are conserved after dedifferentiation and that supernumerary HSA21 has similar effects in the genome-wide dysregulation of gene expression.”
GEDDs are also conserved in syntenic regions of the Ts65Dn mouse model. Ie they are chromosome non-specificially dysregulated.
“The observed conservation also indi- cates that trisomy 21 has a consistent influence on the transcriptome of iPS cells“