Limits...
KLF4 N-terminal variance modulates induced reprogramming to pluripotency.

Kim SI, Oceguera-Yanez F, Hirohata R, Linker S, Okita K, Yamada Y, Yamamoto T, Yamanaka S, Woltjen K - Stem Cell Reports (2015)

Bottom Line: Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms.Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant.Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets.

View Article: PubMed Central - PubMed

Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8507, Japan.

Show MeSH

Related in: MedlinePlus

Gene Expression Patterns Reveal Klf4 Variants(A) Pairwise comparison of global gene expression in mCherry-positive cells on d6 from OSKM, OKMS, and OK+9MS reprogramming. Diagonal lines indicate 2-fold changes in log2 signal intensity. Genes chosen for indexing are highlighted. Green: Krt17, triangle; Sfn, circle; Ocln, square; Cdh1, star; Krt14, inverted triangle. Red: Fut9, triangle; Nr0b1, circle; Cadm1, square; Tbx21, star; Bcl11a, inverted triangle.(B) Left: Venn diagram showing overlapping gene activation versus lacZ-MEFs in the Klf4L cluster. Right: GO analysis of 512 shared d6 genes, arranged in order of p value and indicating the proportion of genes represented for each enriched GO term. Cutoff p = 1.0E-3.(C) Pairwise comparison of global gene expression in mCherry-positive cells on d6 from MKOS and STEMCCA reprogramming. Highlighted genes are those indicated in (A).(D) Dendogram resulting from unsupervised clustering of OSKM, OKMS, OK+9MS, OKN-HAMS, MKOS, STEMCCA, EB-C5, and WTSI mCherry d6 array samples. Segregation of the vectors based on the nature of the KLF4 N terminus is indicated.See also Figure S3 and Table S1.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400650&req=5

fig3: Gene Expression Patterns Reveal Klf4 Variants(A) Pairwise comparison of global gene expression in mCherry-positive cells on d6 from OSKM, OKMS, and OK+9MS reprogramming. Diagonal lines indicate 2-fold changes in log2 signal intensity. Genes chosen for indexing are highlighted. Green: Krt17, triangle; Sfn, circle; Ocln, square; Cdh1, star; Krt14, inverted triangle. Red: Fut9, triangle; Nr0b1, circle; Cadm1, square; Tbx21, star; Bcl11a, inverted triangle.(B) Left: Venn diagram showing overlapping gene activation versus lacZ-MEFs in the Klf4L cluster. Right: GO analysis of 512 shared d6 genes, arranged in order of p value and indicating the proportion of genes represented for each enriched GO term. Cutoff p = 1.0E-3.(C) Pairwise comparison of global gene expression in mCherry-positive cells on d6 from MKOS and STEMCCA reprogramming. Highlighted genes are those indicated in (A).(D) Dendogram resulting from unsupervised clustering of OSKM, OKMS, OK+9MS, OKN-HAMS, MKOS, STEMCCA, EB-C5, and WTSI mCherry d6 array samples. Segregation of the vectors based on the nature of the KLF4 N terminus is indicated.See also Figure S3 and Table S1.

Mentions: Global gene expression has been used to define reprogramming paths. In order to link gene expression to initial reprogramming behaviors, we subjected d6 mCherry+ cells from PB-TAC-OSKM, -OKMS, -OK+9MS, and -MKOS to microarray analysis (Figures 3A and 3B). Based on OSKM and OKMS GO term enrichment (data not shown) and results from previous reports, we established sample indexing criteria (Figure 3A, left). Krt14 and Krt17 (encoding structural proteins), as well as Sfn (a regulator of epithelial cell growth), are claimed to activate in reprogramming populations with preferred iPSC fate (O’Malley et al., 2013). Expression of Ocln and Cdh1 indicates MET responses (Samavarchi-Tehrani et al., 2010), while additional cell-surface changes are reflected by upregulation of Cadm1 and Fut9 (the fucosyltransferase responsible for producing SSEA-1). The developmental regulators Tbx21 and Bcl11a (transiently upregulated non-ESC markers; Polo et al., 2012) and the pre-iPSC marker Nr0b1 (Mikkelsen et al., 2008) show the extent of reprogramming. As early as d6, these ten marker genes were >2-fold differentially expressed between OK+9MS (green series) and OKMS (red series; Figure 3A, left), and <2-fold differentially expressed in a pairwise correlation of OSKM to OK+9MS (R = 0.993; Figure 3A, top right), certifying our indexing for further pairwise comparisons. Correlation and indexing of OSKM to OKMS verified the influence of KLF4 on early gene expression responses (Figure 3A, bottom right).


KLF4 N-terminal variance modulates induced reprogramming to pluripotency.

Kim SI, Oceguera-Yanez F, Hirohata R, Linker S, Okita K, Yamada Y, Yamamoto T, Yamanaka S, Woltjen K - Stem Cell Reports (2015)

Gene Expression Patterns Reveal Klf4 Variants(A) Pairwise comparison of global gene expression in mCherry-positive cells on d6 from OSKM, OKMS, and OK+9MS reprogramming. Diagonal lines indicate 2-fold changes in log2 signal intensity. Genes chosen for indexing are highlighted. Green: Krt17, triangle; Sfn, circle; Ocln, square; Cdh1, star; Krt14, inverted triangle. Red: Fut9, triangle; Nr0b1, circle; Cadm1, square; Tbx21, star; Bcl11a, inverted triangle.(B) Left: Venn diagram showing overlapping gene activation versus lacZ-MEFs in the Klf4L cluster. Right: GO analysis of 512 shared d6 genes, arranged in order of p value and indicating the proportion of genes represented for each enriched GO term. Cutoff p = 1.0E-3.(C) Pairwise comparison of global gene expression in mCherry-positive cells on d6 from MKOS and STEMCCA reprogramming. Highlighted genes are those indicated in (A).(D) Dendogram resulting from unsupervised clustering of OSKM, OKMS, OK+9MS, OKN-HAMS, MKOS, STEMCCA, EB-C5, and WTSI mCherry d6 array samples. Segregation of the vectors based on the nature of the KLF4 N terminus is indicated.See also Figure S3 and Table S1.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400650&req=5

fig3: Gene Expression Patterns Reveal Klf4 Variants(A) Pairwise comparison of global gene expression in mCherry-positive cells on d6 from OSKM, OKMS, and OK+9MS reprogramming. Diagonal lines indicate 2-fold changes in log2 signal intensity. Genes chosen for indexing are highlighted. Green: Krt17, triangle; Sfn, circle; Ocln, square; Cdh1, star; Krt14, inverted triangle. Red: Fut9, triangle; Nr0b1, circle; Cadm1, square; Tbx21, star; Bcl11a, inverted triangle.(B) Left: Venn diagram showing overlapping gene activation versus lacZ-MEFs in the Klf4L cluster. Right: GO analysis of 512 shared d6 genes, arranged in order of p value and indicating the proportion of genes represented for each enriched GO term. Cutoff p = 1.0E-3.(C) Pairwise comparison of global gene expression in mCherry-positive cells on d6 from MKOS and STEMCCA reprogramming. Highlighted genes are those indicated in (A).(D) Dendogram resulting from unsupervised clustering of OSKM, OKMS, OK+9MS, OKN-HAMS, MKOS, STEMCCA, EB-C5, and WTSI mCherry d6 array samples. Segregation of the vectors based on the nature of the KLF4 N terminus is indicated.See also Figure S3 and Table S1.
Mentions: Global gene expression has been used to define reprogramming paths. In order to link gene expression to initial reprogramming behaviors, we subjected d6 mCherry+ cells from PB-TAC-OSKM, -OKMS, -OK+9MS, and -MKOS to microarray analysis (Figures 3A and 3B). Based on OSKM and OKMS GO term enrichment (data not shown) and results from previous reports, we established sample indexing criteria (Figure 3A, left). Krt14 and Krt17 (encoding structural proteins), as well as Sfn (a regulator of epithelial cell growth), are claimed to activate in reprogramming populations with preferred iPSC fate (O’Malley et al., 2013). Expression of Ocln and Cdh1 indicates MET responses (Samavarchi-Tehrani et al., 2010), while additional cell-surface changes are reflected by upregulation of Cadm1 and Fut9 (the fucosyltransferase responsible for producing SSEA-1). The developmental regulators Tbx21 and Bcl11a (transiently upregulated non-ESC markers; Polo et al., 2012) and the pre-iPSC marker Nr0b1 (Mikkelsen et al., 2008) show the extent of reprogramming. As early as d6, these ten marker genes were >2-fold differentially expressed between OK+9MS (green series) and OKMS (red series; Figure 3A, left), and <2-fold differentially expressed in a pairwise correlation of OSKM to OK+9MS (R = 0.993; Figure 3A, top right), certifying our indexing for further pairwise comparisons. Correlation and indexing of OSKM to OKMS verified the influence of KLF4 on early gene expression responses (Figure 3A, bottom right).

Bottom Line: Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms.Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant.Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets.

View Article: PubMed Central - PubMed

Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8507, Japan.

Show MeSH
Related in: MedlinePlus