An autonomous CEBPA enhancer specific for myeloid-lineage priming and neutrophilic differentiation

Avellino, Roberto; Havermans, Marije; Erpelinck, Claudia; Sanders, Mathijs A.; Hoogenboezem, Remco M.; Werken, Harmen J.G. van de; Rombouts, Elwin; Lom, Kirsten van; Strien, Paulette M.H. van; Gebhard, Claudia; Rehli, Michael; Pimanda, John E.; Beck, Dominik; Erkeland, Stefan J.; Kuiken, Thijs; Looper, Hans de; Gröschel, Stefan; Touw, Ivo P.; Bindels, Eric; Delwel, Ruud

doi:10.1182/blood-2016-01-695759

Cited by 61 publications

(77 citation statements)

References 57 publications

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“…4D and Fig. S15) (36). Interestingly, perturbation of this element in the granulocyte/monocyte cell lines HL60 and U937 did not result in any major alteration of CEBPA expression (Fig.…”

Section: Basophil Associations Illuminate Mechanisms For Hematopoieticmentioning

confidence: 77%

“…We next turned to the association with basophil counts at 19q13 near CEBPA. As we noted above, this locus could be resolved to a single putative causal variant, rs78744187, which resides 39 kb downstream from CEBPA, near a separate +42-kb enhancer that has been shown to influence CEBPA expression along various myeloid lineages (36)(37)(38). rs78744187 appeared to be solely associated with basophil counts and showed no evidence of pleiotropic effects on other blood cell traits, including among other myeloid lineages (Dataset S4).…”

Section: Basophil Associations Illuminate Mechanisms For Hematopoieticmentioning

confidence: 95%

“…However, CEBPA is also implicated more broadly in hematopoiesis as a master TF (36,47,48), suggesting that its expression is temporally regulated to specify basophils and other terminal lineages (49). To test whether the +39-kb enhancer provides the temporal regulation of CEBPA expression for proper basophil differentiation, we performed directed differentiation of the CRISPR/Cas9 enhancer-mutagenized HSPCs in the presence of IL-3 (50-52).…”

Section: Basophil Associations Illuminate Mechanisms For Hematopoieticmentioning

confidence: 99%

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Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

Guo

Nandakumar

Ulirsch

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using highcoverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil countassociated locus near the master hematopoietic transcription factor CEBPA. The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance.genome sequencing | GWAS | basophils | hematopoiesis | CEBPA

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“…4D and Fig. S15) (36). Interestingly, perturbation of this element in the granulocyte/monocyte cell lines HL60 and U937 did not result in any major alteration of CEBPA expression (Fig.…”

Section: Basophil Associations Illuminate Mechanisms For Hematopoieticmentioning

confidence: 77%

Section: Basophil Associations Illuminate Mechanisms For Hematopoieticmentioning

confidence: 95%

Section: Basophil Associations Illuminate Mechanisms For Hematopoieticmentioning

confidence: 99%

See 1 more Smart Citation

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

Guo

Nandakumar

Ulirsch

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Indeed, in the last year, several enhancers have been deleted in mice using 2c CRISPR including those associated with expression of Bcl11a , 94 Blimp1 , 95 Mef2c , 96 Stat5 , 97 Pcdh , 98 Mmp13, 99 Gata3 , 100 and Cebpa . 101 In each case, deletion of the enhancer compromised expression of the associated target gene. In addition to enhancers, boundary elements such as locus control regions or insulators may be interrogated with 2c CRISPR.…”

Section: Two-component Crisprmentioning

confidence: 99%

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

Miano

Zhu

Lowenstein

2016

ATVB

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Previous efforts to target the mouse genome for the addition, subtraction, or substitution of biologically informative sequences required complex vector design and a series of arduous steps only a handful of labs could master. The facile and inexpensive clustered regularly interspaced short palindromic repeats (CRISPR) method has now superseded traditional means of genome modification such that virtually any lab can quickly assemble reagents for developing new mouse models for cardiovascular research. Here we briefly review the history of CRISPR in prokaryotes, highlighting major discoveries leading to its formulation for genome modification in the animal kingdom. Core components of CRISPR technology are reviewed and updated. Practical pointers for two-component and three-component CRISPR editing are summarized with a number of applications in mice including frameshift mutations, deletion of enhancers and non-coding genes, nucleotide substitution of protein-coding and gene regulatory sequences, incorporation of loxP sites for conditional gene inactivation, and epitope tag integration. Genotyping strategies are presented and topics of genetic mosaicism and inadvertent targeting discussed. Finally, clinical applications and ethical considerations are addressed as the biomedical community eagerly embraces this astonishing innovation in genome editing to tackle previously intractable questions.

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“…Enhancer activity status depends on the presence of specific histone modifications and other epigenetic marks including cytosine modifications. Enhancers are increasingly shown to play critical roles in myelopoiesis and leukemogenesis (9–11). Recent studies implicate epigenetic enhancer dysfunction through aberrant cytosine methylation as a contributing factor for development of AML in mouse models, for example in the case of Evi1 leukemias (9, 10) or mutant TET2 in combination with FLT3 internal tandem duplications (ITD) (12).…”

Section: Introductionmentioning

confidence: 99%

Epigenetic Identity in AML Depends on Disruption of Nonpromoter Regulatory Elements and Is Affected by Antagonistic Effects of Mutations in Epigenetic Modifiers

et al. 2017

Self Cite

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We performed cytosine methylation sequencing on genetically diverse AML patients and found leukemic DNA methylation patterning is primarily driven by non-promoter regulatory elements and CpG shores. Enhancers displayed stronger differential methylation than promoters, consisting predominantly of hypomethylation. AMLs with dominant hypermethylation featured greater epigenetic disruption of promoters, while those with dominant hypomethylation displayed greater disruption of distal and intronic regions. Mutations in IDH and DNMT3A had opposing and mutually exclusive effects on the epigenome. Notably, co-occurrence of both mutations resulted in epigenetic antagonism, with most CpGs affected by either mutation alone no longer affected in double mutant AMLs. Importantly, this epigenetic antagonism precedes malignant transformation and can be observed in pre-leukemic LSK cells from Idh2R140Q or Dnmt3aR882H single and, Idh2R140Q/Dnmt3aR882H double mutant mice. Notably, IDH/DNMT3A double mutant AMLs manifested upregulation of RAS signaling signature and displayed unique sensitivity to MEK inhibition ex vivo as compared to AMLs with either single mutation.

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An autonomous CEBPA enhancer specific for myeloid-lineage priming and neutrophilic differentiation

Abstract: Key Points The CEBPA locus harbors 14 enhancers of which distinct combinations are active in different CEBPA-expressing tissues. A +42-kb enhancer is required for myeloid-lineage priming to drive adequate CEBPA expression levels necessary for neutrophilic maturation.

Cited by 61 publications

References 57 publications

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

Epigenetic Identity in AML Depends on Disruption of Nonpromoter Regulatory Elements and Is Affected by Antagonistic Effects of Mutations in Epigenetic Modifiers

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