A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation
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eLife assessment
Authors provide valuable evidence identifying a lncRNA transcribed specifically in the pDC subtype from the +32Kb promoter region which is also the region for the enhancer for Irf8 specifically in the cDC1 subtype. With convincing methodology, they provide in-depth analysis about the possible role of lncIrf8, and its promoter region and cross-talk with Irf8 promoter to identify that it is not the lncIRF8 itself but its promoter region that is crucial for pDC and cDC1 differentiation conferring feedback inhibition of Irf8 transcription. The work will be of interest to immunologists working on immune cell development.
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Abstract
Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8) is a lineage determining transcription factor in hematopoiesis and master regulator of dendritic cells (DC), an important immune cell for immunity and tolerance. IRF8 is prominently upregulated in DC development by autoactivation and controls both DC differentiation and function. However, it is unclear how Irf8 autoactivation is controlled and eventually limited. Here, we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of Irf8 transcription start site and expressed specifically in mouse plasmacytoid DC (pDC), referred to as lncIrf8 . The lncIrf8 locus interacts with the lrf8 promoter and shows differential epigenetic signatures in pDC versus classical DC type 1 (cDC1). Interestingly, a sequence element of the lncIrf8 promoter, but not lncIrf8 itself, is crucial for mouse pDC and cDC1 differentiation, and this sequence element confers feedback inhibition of Irf8 expression. Taken together, in DC development Irf8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIrf8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of Irf8 that orchestrates its own expression and thereby controls DC differentiation.
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Author Response
Reviewer #1 (Public Review):
IRF8 is a key transcription factor in the differentiation of hematopoietic cell lineages including dendritic cell (DC) and monocyte/macrophage lineages. The promoter and enhancer regions of Irf8 have been a focus of intense research in recent times. In the submitted study Xu H. et. Al., have first time reported a lncRNA transcribed specifically in the pDC subtype from +32Kb which is also the region for the enhancer for Irf8 specifically in the cDC1 subtype. Authors have employed modern-day tools for an in-depth understanding of the role of lncIrf8, its promoter region, and crosstalk with Irf8 promoter to identify that it is not the lncIRF8 itself but its promoter region is crucial for pDC and cDC1 differentiation conferring feedback inhibition of Irf8 transcription. In the attempt to …
Author Response
Reviewer #1 (Public Review):
IRF8 is a key transcription factor in the differentiation of hematopoietic cell lineages including dendritic cell (DC) and monocyte/macrophage lineages. The promoter and enhancer regions of Irf8 have been a focus of intense research in recent times. In the submitted study Xu H. et. Al., have first time reported a lncRNA transcribed specifically in the pDC subtype from +32Kb which is also the region for the enhancer for Irf8 specifically in the cDC1 subtype. Authors have employed modern-day tools for an in-depth understanding of the role of lncIrf8, its promoter region, and crosstalk with Irf8 promoter to identify that it is not the lncIRF8 itself but its promoter region is crucial for pDC and cDC1 differentiation conferring feedback inhibition of Irf8 transcription. In the attempt to decipher the crosstalk between the promoter regions of IRF8 and lncIRF8 by employing various in vitro artificial systems, the study falls short of identifying the real significance of the lncIRF8 which is specifically expressed in pDC subtype.
We appreciate the public review made by the reviewer. We agree with the reviewer that most of the experiments on the identification of the negative feedback regulation of IRF8 via the lncIRF8 promoter element were carried out in vitro. But we would like to point out also our in vivo work: (i) transplantation lncIRF8 promoter KO cells into mice demonstrates that pDC and cDC1 development were compromised (Figure 3); (ii) lncIRF8 is expressed in in vivo BM and spleen pDC (new Figure 1-figure supplement 3). We also would like to emphasize that (i) in vivo studies on the identification of the negative feedback regulation of IRF8 via the lncIRF8 promoter element and (ii) mechanistic studies with CRISPR activation and CRISPR interference would have been difficult to perform in vivo with current tools available in mice.
According to our current understand lncIRF8 act as an indicator of +32 kb enhancer activity and we agree with the reviewer that further potential functions of lncIRF8 still need to be explored. We added a sentence on page 13, lines 427 and 428 on potential additional functions of lncIRF8:
"However, lncIRF8 might have additional functions in DC biology, which are not revealed in the current study and remain to be identified."
Reviewer #2 (Public Review):
The manuscript of Xu and colleagues examines in detail the regulation of the important transcription factor IRF8 in dendritic cell (DC) subsets. They identify a long noncoding RNA arises from the +32kb enhancer of IRF8 specifically in plasmacytoid DCs (pDCs)and show clearly that this lncIRF8 marks the activity of a region of this enhancer but the RNA itself does not appear to have any function. Deletion of the promoter of the lncIRF8 ablated cDC1 and pDC differentiation using an in vitro cell differentiation model. The authors propose an innovative model that the lncIRF8 promoter sequences act to limit IRF8 expression in cDC1, but are inactive in pDCs, resulting in their characteristically very high IRF8 expression.
This is a conceptually interesting study that makes excellent use of an extensive set of genomic data for the DC subsets. There has been a lot of recent research investigating the regulation of the IRF8 gene in hematopoiesis and this study provides an important new aspect to the work. The use of an in vitro model of DC differentiation is a powerful practical approach to investigating IRF8 regulation, as is the innovative use of CRISPR technology. Perhaps the biggest limitation of this study is that the authors have not conformed to the in-cell system data by creating a mouse strain lacking the lncIRF8 element. Such approaches by others, most notably the Murphy lab, have been instrumental in pushing this field forward. Nevertheless, Xu et al. significantly add to our current knowledge of the regulation of IRF8, a critical step in forming the dendritic cell network.
We appreciate the public review made by the reviewer and the positive assessment of our work. We agree with the review that extending our in-cell system data to lncIRF8 promoter KO mice will further strengthen our data and this will be subject of our future work.
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eLife assessment
Authors provide valuable evidence identifying a lncRNA transcribed specifically in the pDC subtype from the +32Kb promoter region which is also the region for the enhancer for Irf8 specifically in the cDC1 subtype. With convincing methodology, they provide in-depth analysis about the possible role of lncIrf8, and its promoter region and cross-talk with Irf8 promoter to identify that it is not the lncIRF8 itself but its promoter region that is crucial for pDC and cDC1 differentiation conferring feedback inhibition of Irf8 transcription. The work will be of interest to immunologists working on immune cell development.
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Reviewer #1 (Public Review):
IRF8 is a key transcription factor in the differentiation of hematopoietic cell lineages including dendritic cell (DC) and monocyte/macrophage lineages. The promoter and enhancer regions of Irf8 have been a focus of intense research in recent times. In the submitted study Xu H. et. Al., have first time reported a lncRNA transcribed specifically in the pDC subtype from +32Kb which is also the region for the enhancer for Irf8 specifically in the cDC1 subtype. Authors have employed modern-day tools for an in-depth understanding of the role of lncIrf8, its promoter region, and crosstalk with Irf8 promoter to identify that it is not the lncIRF8 itself but its promoter region is crucial for pDC and cDC1 differentiation conferring feedback inhibition of Irf8 transcription. In the attempt to decipher the crosstalk …
Reviewer #1 (Public Review):
IRF8 is a key transcription factor in the differentiation of hematopoietic cell lineages including dendritic cell (DC) and monocyte/macrophage lineages. The promoter and enhancer regions of Irf8 have been a focus of intense research in recent times. In the submitted study Xu H. et. Al., have first time reported a lncRNA transcribed specifically in the pDC subtype from +32Kb which is also the region for the enhancer for Irf8 specifically in the cDC1 subtype. Authors have employed modern-day tools for an in-depth understanding of the role of lncIrf8, its promoter region, and crosstalk with Irf8 promoter to identify that it is not the lncIRF8 itself but its promoter region is crucial for pDC and cDC1 differentiation conferring feedback inhibition of Irf8 transcription. In the attempt to decipher the crosstalk between the promoter regions of IRF8 and lncIRF8 by employing various in vitro artificial systems, the study falls short of identifying the real significance of the lncIRF8 which is specifically expressed in pDC subtype.
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Reviewer #2 (Public Review):
The manuscript of Xu and colleagues examines in detail the regulation of the important transcription factor IRF8 in dendritic cell (DC) subsets. They identify a long noncoding RNA arises from the +32kb enhancer of IRF8 specifically in plasmacytoid DCs (pDCs)and show clearly that this lncIRF8 marks the activity of a region of this enhancer but the RNA itself does not appear to have any function. Deletion of the promoter of the lncIRF8 ablated cDC1 and pDC differentiation using an in vitro cell differentiation model. The authors propose an innovative model that the lncIRF8 promoter sequences act to limit IRF8 expression in cDC1, but are inactive in pDCs, resulting in their characteristically very high IRF8 expression.
This is a conceptually interesting study that makes excellent use of an extensive set of …
Reviewer #2 (Public Review):
The manuscript of Xu and colleagues examines in detail the regulation of the important transcription factor IRF8 in dendritic cell (DC) subsets. They identify a long noncoding RNA arises from the +32kb enhancer of IRF8 specifically in plasmacytoid DCs (pDCs)and show clearly that this lncIRF8 marks the activity of a region of this enhancer but the RNA itself does not appear to have any function. Deletion of the promoter of the lncIRF8 ablated cDC1 and pDC differentiation using an in vitro cell differentiation model. The authors propose an innovative model that the lncIRF8 promoter sequences act to limit IRF8 expression in cDC1, but are inactive in pDCs, resulting in their characteristically very high IRF8 expression.
This is a conceptually interesting study that makes excellent use of an extensive set of genomic data for the DC subsets. There has been a lot of recent research investigating the regulation of the IRF8 gene in hematopoiesis and this study provides an important new aspect to the work. The use of an in vitro model of DC differentiation is a powerful practical approach to investigating IRF8 regulation, as is the innovative use of CRISPR technology. Perhaps the biggest limitation of this study is that the authors have not conformed to the in-cell system data by creating a mouse strain lacking the lncIRF8 element. Such approaches by others, most notably the Murphy lab, have been instrumental in pushing this field forward. Nevertheless, Xu et al. significantly add to our current knowledge of the regulation of IRF8, a critical step in forming the dendritic cell network.
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