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Changes in expression of the long non-coding RNA FMR4 associate with altered gene expression during differentiation of human neural precursor cells.

Peschansky VJ, Pastori C, Zeier Z, Motti D, Wentzel K, Velmeshev D, Magistri M, Bixby JL, Lemmon VP, Silva JP, Wahlestedt C - Front Genet (2015)

Bottom Line: Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays.Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4.Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

View Article: PubMed Central - PubMed

Affiliation: Center for Therapeutic Innovation and Department of Psychiatry and Behavioral Sciences, Miller School of Medicine, University of Miami Miami, FL, USA.

ABSTRACT
CGG repeat expansions in the Fragile X mental retardation 1 (FMR1) gene are responsible for a family of associated disorders characterized by either intellectual disability and autism Fragile X Syndrome (FXS), or adult-onset neurodegeneration Fragile X-associated Tremor/Ataxia Syndrome. However, the FMR1 locus is complex and encodes several long non-coding RNAs, whose expression is altered by repeat expansion mutations. The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4, which we previously identified. "Full"-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4, thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays. We identified FMR4-responsive genes, including the methyl-CpG-binding domain protein 4 (MBD4). Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4. We therefore propose that FMR4's function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

No MeSH data available.


Related in: MedlinePlus

Microarray analysis of mRNA expression changes in response to FMR4 knockdown and overexpression reveal trans-regulatory targets. (A) Microarray analysis of HEK293T cells with FMR4 overexpression or knockdown (n = 3). LIMMA analysis and Cluster Affinity identified genes concordantly or discordantly regulated compared to FMR4. (B)FMR4 was knocked down with siRNA in HEK293T cells in order to validate changes in a putative FMR4-sensitive gene identified by microarray (C). (n = 6, *p < 0.05 by Student’s t-test).
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Figure 1: Microarray analysis of mRNA expression changes in response to FMR4 knockdown and overexpression reveal trans-regulatory targets. (A) Microarray analysis of HEK293T cells with FMR4 overexpression or knockdown (n = 3). LIMMA analysis and Cluster Affinity identified genes concordantly or discordantly regulated compared to FMR4. (B)FMR4 was knocked down with siRNA in HEK293T cells in order to validate changes in a putative FMR4-sensitive gene identified by microarray (C). (n = 6, *p < 0.05 by Student’s t-test).

Mentions: Previous studies of the 2.4 kb antisense lncRNA FMR4 described no cis-regulation of FMR1 (Khalil et al., 2008); therefore we hypothesized that FMR4 would regulate gene expression in trans, which is a well-documented function of other lncRNAs (Rinn et al., 2007; Kino et al., 2010; Miyagawa et al., 2012). In order to comprehensively measure gene regulation in response to FMR4 at the mRNA level, we treated HEK293T cells with either an siRNA against FMR4, a scrambled control siRNA (knockdown), pcDNA3.1-FMR4, or the empty pcDNA3.1 vector (overexpression) and processed for microarray hybridization after 6 or 24 h. Using LIMMA, a linear modeling approach (Smyth, 2004), we identified differential expression of over 3,700 genes between FMR4 knockdown, overexpression and their respective control conditions, and characterized the pattern of target gene expression relative to FMR4. To this end, we used the Cluster Affinity tool of TIGR MultiExperiment Viewer to identify genes with opposite behavior in the knockdown condition compared to the overexpression condition. This strategy narrowed our focus to the 238 transcripts represented in Figure 1A and Supplementary Figure S1, which are further classified by whether they are concordant or discordant with respect to FMR4 changes. This analysis yielded 155 and 83 target genes with concordant and discordant changes in mRNA expression relative to FMR4, respectively. These data support the idea that FMR4 is a regulator of gene expression through trans-activity.


Changes in expression of the long non-coding RNA FMR4 associate with altered gene expression during differentiation of human neural precursor cells.

Peschansky VJ, Pastori C, Zeier Z, Motti D, Wentzel K, Velmeshev D, Magistri M, Bixby JL, Lemmon VP, Silva JP, Wahlestedt C - Front Genet (2015)

Microarray analysis of mRNA expression changes in response to FMR4 knockdown and overexpression reveal trans-regulatory targets. (A) Microarray analysis of HEK293T cells with FMR4 overexpression or knockdown (n = 3). LIMMA analysis and Cluster Affinity identified genes concordantly or discordantly regulated compared to FMR4. (B)FMR4 was knocked down with siRNA in HEK293T cells in order to validate changes in a putative FMR4-sensitive gene identified by microarray (C). (n = 6, *p < 0.05 by Student’s t-test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4530595&req=5

Figure 1: Microarray analysis of mRNA expression changes in response to FMR4 knockdown and overexpression reveal trans-regulatory targets. (A) Microarray analysis of HEK293T cells with FMR4 overexpression or knockdown (n = 3). LIMMA analysis and Cluster Affinity identified genes concordantly or discordantly regulated compared to FMR4. (B)FMR4 was knocked down with siRNA in HEK293T cells in order to validate changes in a putative FMR4-sensitive gene identified by microarray (C). (n = 6, *p < 0.05 by Student’s t-test).
Mentions: Previous studies of the 2.4 kb antisense lncRNA FMR4 described no cis-regulation of FMR1 (Khalil et al., 2008); therefore we hypothesized that FMR4 would regulate gene expression in trans, which is a well-documented function of other lncRNAs (Rinn et al., 2007; Kino et al., 2010; Miyagawa et al., 2012). In order to comprehensively measure gene regulation in response to FMR4 at the mRNA level, we treated HEK293T cells with either an siRNA against FMR4, a scrambled control siRNA (knockdown), pcDNA3.1-FMR4, or the empty pcDNA3.1 vector (overexpression) and processed for microarray hybridization after 6 or 24 h. Using LIMMA, a linear modeling approach (Smyth, 2004), we identified differential expression of over 3,700 genes between FMR4 knockdown, overexpression and their respective control conditions, and characterized the pattern of target gene expression relative to FMR4. To this end, we used the Cluster Affinity tool of TIGR MultiExperiment Viewer to identify genes with opposite behavior in the knockdown condition compared to the overexpression condition. This strategy narrowed our focus to the 238 transcripts represented in Figure 1A and Supplementary Figure S1, which are further classified by whether they are concordant or discordant with respect to FMR4 changes. This analysis yielded 155 and 83 target genes with concordant and discordant changes in mRNA expression relative to FMR4, respectively. These data support the idea that FMR4 is a regulator of gene expression through trans-activity.

Bottom Line: Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays.Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4.Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

View Article: PubMed Central - PubMed

Affiliation: Center for Therapeutic Innovation and Department of Psychiatry and Behavioral Sciences, Miller School of Medicine, University of Miami Miami, FL, USA.

ABSTRACT
CGG repeat expansions in the Fragile X mental retardation 1 (FMR1) gene are responsible for a family of associated disorders characterized by either intellectual disability and autism Fragile X Syndrome (FXS), or adult-onset neurodegeneration Fragile X-associated Tremor/Ataxia Syndrome. However, the FMR1 locus is complex and encodes several long non-coding RNAs, whose expression is altered by repeat expansion mutations. The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4, which we previously identified. "Full"-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4, thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays. We identified FMR4-responsive genes, including the methyl-CpG-binding domain protein 4 (MBD4). Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4. We therefore propose that FMR4's function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

No MeSH data available.


Related in: MedlinePlus