Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity

Abstract

RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both). RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both). Type 3 RNA polymerase (Pol III) promoters, such as 7SK, U6, and H1, are popular for the expression of small noncoding RNAs because of their robust level of transcription in all cell types and defined transcription initiation and termination sites, such that a precise (therapeutic) transcript is made (1Kole R. Krainer A.R. Altman S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides.Nat. Rev. Drug Discov. 2012; 11: 125-140Crossref PubMed Scopus (852) Google Scholar, 2Schramm L. Hernandez N. Recruitment of RNA polymerase III to its target promoters.Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (448) Google Scholar). These promoters are unique in that all critical elements are located upstream of the transcriptional initiation site, thus enabling the expression of almost any small RNA sequence, including siRNA or shRNA for RNAi purposes, and guide RNA for CRISPR-mediated genome editing applications (1Kole R. Krainer A.R. Altman S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides.Nat. Rev. Drug Discov. 2012; 11: 125-140Crossref PubMed Scopus (852) Google Scholar, 2Schramm L. Hernandez N. Recruitment of RNA polymerase III to its target promoters.Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (448) Google Scholar, 3Fellmann C. Lowe S.W. Stable RNA interference rules for silencing.Nat. Cell Biol. 2014; 16: 10-18Crossref PubMed Scopus (140) Google Scholar, 4Kabadi A.M. Ousterout D.G. Hilton I.B. Gersbach C.A. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.Nucleic Acids Res. 2014; 42: e147Crossref PubMed Scopus (233) Google Scholar). We previously reported that these Pol III promoters can also load Pol II and synthesize lengthy translation-competent mRNAs (5Gao Z. Herrera-Carrillo E. Berkhout B. RNA polymerase II activity of type 3 Pol III promoters.Mol. Ther. Nucleic Acids. 2018; 12: 135-145Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). These three promoters differ profoundly in Pol II strength (H1 >> U6 > 7SK). Pol II strength of H1 is higher than that of the commonly used simian virus 40 (SV40) early Pol II promoter, whereas 7SK exhibits only minimal Pol II activity. We previously estimated a 3-fold ratio of Pol III to Pol II transcription by the H1 promoter (5Gao Z. Herrera-Carrillo E. Berkhout B. RNA polymerase II activity of type 3 Pol III promoters.Mol. Ther. Nucleic Acids. 2018; 12: 135-145Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Thus, among these three promoters, 7SK is the promoter of choice in terms of Pol III specificity, whereas H1 can hardly be considered a Pol III-specific promoter. This dual activity of the H1 promoter does however provide an ingenious possibility for the simultaneous expression of both a small noncoding RNA and an mRNA. We explored this unique feature to coexpress both a guide RNA and the Cas9 endonuclease, thus constituting a single promoter-driven CRISPR–Cas9 system that provides a significant titer advantage in lentiviral vector production (6Gao Z. Herrera-Carrillo E. Berkhout B. A single H1 promoter can drive both guide RNA and endonuclease expression in the CRISPR-Cas9 system.Mol. Ther. Nucleic Acids. 2019; 14: 32-40Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). We also envisioned that one could modulate the Pol II/III ratio to further broaden the applicability of the H1 system. In a previous study, we showed that mutation of the TATA box of type 3 RNA Pol III promoters, including H1, abolished Pol III activity and enhanced Pol II activity, the latter presumably caused by the loss of competitive Pol III binding to the promoter (5Gao Z. Herrera-Carrillo E. Berkhout B. RNA polymerase II activity of type 3 Pol III promoters.Mol. Ther. Nucleic Acids. 2018; 12: 135-145Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The reverse strategy, which is to construct a Pol III-specific H1 promoter, is also of interest as this would not only boost the expression of the small RNA but also—perhaps more importantly—disrupt the generation of undesired and lengthy Pol II transcripts that may even encode an immunogenic protein, a scenario that is to be avoided, especially in clinical gene therapy settings. The profound difference in Pol II activity of the H1 and 7SK promoters is quite remarkable as they share a similar type 3 RNA Pol III promoter architecture (7Myslinski E. Ame J.C. Krol A. Carbon P. An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene.Nucleic Acids Res. 2001; 29: 2502-2509Crossref PubMed Scopus (128) Google Scholar). Both promoters consist of a basal region directing basal transcription and a distal sequence element (DSE) that enhances transcription (2Schramm L. Hernandez N. Recruitment of RNA polymerase III to its target promoters.Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (448) Google Scholar) (Fig. 1A). The basal region is composed of a proximal sequence element (PSE) and a TATA box that are separated by a spacer. The DSE encodes an octamer motif and a Staf binding site (Staf). However, the relative position and exact nucleotide sequence of these elements differ between H1 and 7SK (7Myslinski E. Ame J.C. Krol A. Carbon P. An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene.Nucleic Acids Res. 2001; 29: 2502-2509Crossref PubMed Scopus (128) Google Scholar). For example, these elements are distributed over approximately 240 bp of the 7SK promoter, whereas they lie within a compact 100-bp region of the H1 promoter. Based on the overall similarity in promoter architecture, we hypothesized that it would be possible to engineer a Pol III-specific H1 promoter by replacing H1 elements by corresponding 7SK motifs. To this end, we first delineated which sequences of the H1 promoter are critical for Pol II/III activity and specificity. By systematic swapping of elements, we engineered a compact H1/7SK hybrid promoter with enhanced Pol III activity and specificity. In parallel, we also performed an in-depth analysis of two Pol II-specific H1 variants with a mutated TATA box and explored their potential as general Pol II promoters for protein expression. A previous H1 promoter study showed that the important Pol III transcription elements are located within 100 bp (H1-100) upstream of the transcription initiation site (7Myslinski E. Ame J.C. Krol A. Carbon P. An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene.Nucleic Acids Res. 2001; 29: 2502-2509Crossref PubMed Scopus (128) Google Scholar). the H1 promoter that is used for small RNA expression is A.M. Ousterout D.G. Hilton I.B. Gersbach C.A. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.Nucleic Acids Res. 2014; 42: e147Crossref PubMed Scopus (233) Google Scholar, Berkhout B. vector for shRNA expression and Ther. 16: Full Text Full Text PDF PubMed Scopus Google Scholar). The elements for Pol III activity of the H1 promoter are but these elements are also for the Pol II activity To we first delineated the H1 for Pol II and III activity. The basal region the with the and TATA box is to be for We of bp in the H1 promoter, a of promoter variants (Fig. and of To the promoter the H1 variants to both a small RNA by Pol III and a by Pol II (Fig. 1A). of the human and cell Pol II activity by and Pol III activity by a for the The short transcript by the and similar expression to construct but expression for of is in This that the sequence between position and is critical for Pol III activity. To the critical elements in more we in The with to Pol III transcriptional activity of is in but the promoter variants either and or all and Pol III activity. The from to the octamer motif position to (Fig. thus the of this DSE for Pol III These are also in with the that as the minimal Pol III promoter (7Myslinski E. Ame J.C. Krol A. Carbon P. An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene.Nucleic Acids Res. 2001; 29: 2502-2509Crossref PubMed Scopus (128) Google Scholar). the Pol II activity on for the of H1 promoters showed a similar as the Pol III activity (Fig. The first variants and the Pol II promoter activity is in such that is and more than and The Pol II activity to a level that of for the in to of the octamer in and Pol II even to a than the in Pol III Full Pol II is for and The overall Pol II/III activity are thus quite that Pol II and III may use the promoter elements and that the octamer is critical for both In these that to as the the minimal sequences for both Pol II and III activity. The promoter is attractive for applications vector that a small Pol III expression However, its promiscuous Pol II activity is and can be in the of a RNA or an that is an and immunogenic We to engineer a Pol III-specific promoter. is similarity between the H1 and 7SK promoters of but the latter Pol II activity. We that swapping of promoter elements from 7SK H1 may Pol III the promoter. We thus by or 7SK elements TATA and the between the latter two and the Pol II/III activity We these and the Pol II and III activity by and The profoundly in which the Pol III with the parental promoter (Fig. is in We three hybrid with Pol III activity and in which the showed a that the 7SK Pol III transcription to a than the H1 this not the Pol II activity, that Pol II to III is not the for the Pol III activity. and 7SK elements and and the Pol III activity to an even than and Pol II activity of the also and not to the Pol III activity (Fig. Pol II activity with for that similar activity and that showed activity. The robust in Pol II activity on of elements and with the by The construct is the Pol III-specific promoter, the Pol III activity with a profoundly Pol II activity. We that in which H1 elements and TATA more than of its sequence from may thus be to a hybrid H1/7SK promoter, this is a are in In is the promoter of choice for Pol III applications because of its Pol III activity and minimal Pol II activity. To applications in it is important that this hybrid promoter in cell with a of transcription To we the Pol III activity of with and two popular Pol III promoters and in three cell of The cell and and RNA for analysis (Fig. is in of We a similar Pol III activity in these cell the and promoters similar Pol III activity, which is in and especially in the promoter is more than the two parental and 7SK We performed to that the Pol II activity of and is a general feature of these activity (Fig. (Fig. and (Fig. A similar activity in all cell and Pol II activity, which is for and especially that Pol II activity as the 7SK promoter (5Gao Z. Herrera-Carrillo E. Berkhout B. RNA polymerase II activity of type 3 Pol III promoters.Mol. Ther. Nucleic Acids. 2018; 12: 135-145Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We that the enhanced Pol III activity and of the H1/7SK hybrid promoter does not to on the cell of the and robust Pol II activity of the small H1 promoter, we to study its potential as promoter of choice for is the Pol II but the promoter, which is than the also activity. In further of the Pol II activity, we that of the TATA box would not only Pol III activity but also boost Pol II activity as we reported for (5Gao Z. Herrera-Carrillo E. Berkhout B. RNA polymerase II activity of type 3 Pol III promoters.Mol. Ther. Nucleic Acids. 2018; 12: 135-145Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We the TATA box and and the Pol II activity. We the of H1 in cell types and and activity (Fig. The widely the cell but a general for the H1 We that is more than in with the in (Fig. Both and showed enhanced Pol II activity with the parental promoters in of the cell to of the TATA box enhanced Pol II activity to a in than in but than and Thus, is the H1 with the Pol II activity, whereas is the promoter of choice the promoter size a To a of the Pol II activity, we the H1 variants to Pol II promoters and The size of these promoters is in the significant size advantage of The and promoters the expression level the cell and are also the is to than the promoter but exhibits similar Pol II activity as early is the promoter. the small promoter is in Pol II activity to and thereby that is a Pol II promoter that level Pol II and of the promoters used in this R. The transcriptional site for a human U6 small RNA gene is by a promoter element of the and the TATA Acids Res. PubMed Scopus Google Z. Herrera-Carrillo E. Berkhout B. A single H1 promoter can drive both guide RNA and endonuclease expression in the CRISPR-Cas9 system.Mol. Ther. Nucleic Acids. 2019; 14: 32-40Abstract Full Text Full Text PDF PubMed Scopus (19) Google A.M. human U6 small RNA with transcriptional Acids Res. PubMed Scopus Google simian virus in a simian virus gene therapy applications use that the human we the activity of the H1 promoters in the (Fig. expression and cell The construct because the sequences are in the lentiviral vector which may lentiviral vector production and Berkhout B. vector for shRNA expression and Ther. 16: Full Text Full Text PDF PubMed Scopus Google Scholar). We used a of of for lentiviral to a single The by The in are with the (Fig. that is the H1 the cell H1 variants activity than the promoter and are in strength to the and In the and promoters robust Pol II activity that is in a that both promoters could the promoter of choice for Pol II the advantage of but would be the size of the is a in and are in The H1 promoter is a type 3 RNA Pol III promoter and is popular for the expression of small noncoding RNAs (1Kole R. Krainer A.R. Altman S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides.Nat. Rev. Drug Discov. 2012; 11: 125-140Crossref PubMed Scopus (852) Google Scholar, 2Schramm L. Hernandez N. Recruitment of RNA polymerase III to its target promoters.Genes Dev. 2002; 16: 2593-2620Crossref PubMed Scopus (448) Google Scholar). that type 3 Pol III promoters can also a level of Pol II transcription and that the H1 promoter unusually Pol II activity (5Gao Z. Herrera-Carrillo E. Berkhout B. RNA polymerase II activity of type 3 Pol III promoters.Mol. Ther. Nucleic Acids. 2018; 12: 135-145Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In this study, we that the promoter is the minimal for both Pol II and Pol III activity. Pol III activity of is to that of the promoter, whereas Pol II activity is The dual Pol II/III activity of the H1 promoter is an undesired feature in For example, in gene applications the H1 promoter is used to short shRNA the could be as both Pol II and Pol III transcripts the hairpin that can to form an between the two In this the Pol II transcript can with the and of the short Pol III shRNA In Pol II transcription from the H1 promoter may Pol II transcription and of the polymerase or transcriptional interference RNA polymerase II 2012; Full Text Full Text PDF PubMed Scopus Google Scholar, interference by RNA polymerase and of transcription PubMed Scopus Google Scholar). an is located of the H1 promoter, the Pol II transcript can be a protein with or immunogenic a scenario that especially be in a gene therapy an can the of the E. of gene therapy for PubMed Scopus Google Scholar, C. S. Z. P. C. C. The potential of the gene does not clinical with the use of in for PubMed Scopus Google Scholar, A. not to be in gene Ther. PubMed Scopus Google Scholar). potential can more Pol III promoters are for applications A.M. Ousterout D.G. Hilton I.B. Gersbach C.A. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.Nucleic Acids Res. 2014; 42: e147Crossref PubMed Scopus (233) Google Scholar, Berkhout B. vector for shRNA expression and Ther. 16: Full Text Full Text PDF PubMed Scopus Google Scholar). dual promoter activity is we to Pol Pol III-specific H1 of a Pol III-specific H1 promoter by replacing H1 promoter elements by of 7SK, a popular type 3 Pol III promoter with Pol III (5Gao Z. Herrera-Carrillo E. Berkhout B. RNA polymerase II activity of type 3 Pol III promoters.Mol. Ther. Nucleic Acids. 2018; 12: 135-145Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). of the basal region and TATA with the 7SK octamer the H1/7SK hybrid promoter with minimal Pol II activity and Pol III activity with both parental The promoter is a to the Pol III that we for Pol III applications of H1 or of to of gene in which its promoter. The use of promoters is not as sequence are to especially in Berkhout B. vector for shRNA expression and Ther. 16: Full Text Full Text PDF PubMed Scopus Google Scholar). with promoters be used Berkhout B. vector for shRNA expression and Ther. 16: Full Text Full Text PDF PubMed Scopus Google Scholar). For the Pol III-specific promoter could be with 7SK as the sequence is also provide the basal of 7SK is for Pol III activity and specificity. promoters in two with or a TATA box Hernandez N. to the RNA from 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). with both a and TATA box Pol III whereas promoters Pol II In Pol II the protein the and both the binding protein and the transcription that Pol II protein and binding protein are also to promoters with a and TATA but or is the of and with Pol Thus, Pol III are to both the and TATA box and the between these elements, which the (Fig. The is critical and from to Hernandez N. study of RNA polymerase II and III promoters a gene by both and a use of 2012; PubMed Scopus Google Scholar). or of bp the U6 Pol III promoter R. The transcriptional site for a human U6 small RNA gene is by a promoter element of the and the TATA Acids Res. PubMed Scopus Google Scholar) and that the and TATA box are of Pol II/III specificity. experiments demonstrated that promoters TATA almost Pol whereas with a TATA box Pol III Hernandez N. study of RNA polymerase II and III promoters a gene by both and a use of 2012; PubMed Scopus Google Scholar). The H1 promoter is in that it both Pol II and the is only the H1 promoter both Pol II and Pol III with other promoters, two H1 H1 the TATA box with and and H1 the spacer. all a scenario in which the in the H1 promoter is for of the and TATA box (Fig. the is only to the but not the TATA Pol II expression is similar to We thus that the is a critical of Pol III specificity. In with this is that all 7SK Pol II activity. the Pol III activity of and especially in which the or and TATA box by 7SK elements, one may higher Pol III activity. of the sequences provides a possible The H1 that we is (7Myslinski E. Ame J.C. Krol A. Carbon P. An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene.Nucleic Acids Res. 2001; 29: 2502-2509Crossref PubMed Scopus (128) Google Scholar, Hernandez N. study of RNA polymerase II and III promoters a gene by both and a use of 2012; PubMed Scopus Google whereas the of is A.M. human U6 small RNA with transcriptional Acids Res. PubMed Scopus Google Scholar, S.W. R. 7SK promoter for short hairpin RNA 2012; Scholar). The H1 element to the but the of the H1 is in a This the defined H1 sequence is To we mutated this and Pol II and Pol III activity This that and which the sequence, a in which may their Pol III activity. of the may also that the of Pol II/III is even more For example, in we the H1 by that of 7SK and the H1 and TATA This exhibits Pol III over the parental H1 promoter by Pol III activity. Thus, the 7SK element higher Pol III activity and with the H1 in which we the H1 TATA box is also as the 7SK TATA box enhances promoter activity, not specificity, which may also to the enhanced Pol III activity of and In we that the basal 7SK region is for Pol III for three the Pol III of the 7SK the 7SK for binding to the and TATA and the 7SK TATA box that enhances Pol II and III activity. To engineer Pol II-specific H1 promoters, we mutated the TATA box of two H1 size and and these promoters and We previously used this TATA to the H1 promoter Pol II (5Gao Z. Herrera-Carrillo E. Berkhout B. RNA polymerase II activity of type 3 Pol III promoters.Mol. Ther. Nucleic Acids. 2018; 12: 135-145Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Both H1 variants Pol II activity that is similar to that of the commonly used Pol II promoters and is than the size of the gene construct is an important the bp promoter an attractive The Pol II-specific H1 promoter may over Pol II promoters for gene expression the Pol II-specific is only bp in which the size of but is than commonly used Pol II promoters with a size of bp is even and among the Pol II promoters, including such as the promoter A. S. R. promoters for PubMed Scopus Google Scholar). The size of these two H1 promoter variants may provide a advantage in a is by with a This is for in lentiviral that the Cas9 endonuclease and a of the size of the Cas9 small promoters such as are used for its expression. a to be one can use site but this in a expression of the may thus be that the its promoter. such size are a small and Pol II promoter or is that the Pol II promoter activity of and is of the cell This may as the of the H1 promoter is to drive the expression of the RNA of the in in all cell types C. Altman S. and transcription of a human gene for H1 the RNA of human Acids Res. PubMed Scopus Google Scholar). In other the Pol III activity of the H1 promoter is in all but we not the of the Pol II activity. we demonstrated that the Pol II-specific promoter is in strength to the commonly used and promoters and that is only a Both promoters thus Pol II activity to be considered as general Pol II we the Pol III activity of and whereas of the used Pol II promoters may Pol III activity. such activity not it may as small transcripts not to the of expression of RNA transcripts by Pol III promoters, one can that short RNA expression by Pol II promoters may cell types and are to transcriptional or promoter and the use of the promoter is not for this Pol II-specific promoters are of interest as their Pol III may to than Pol II This would for the H1 promoter as is a for the H1 transcript of the Thus, especially for gene therapy it would be important to the of and gene expression. 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Nucleic Acids. 2019; 14: 32-40Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). in to a of approximately the of The of lentiviral with a of and The by of The and of performed by to the titer of the For expression from a of of used for of and activity from an position with lentiviral that which by with cell with and performed by an with the cell system. The of by are within the The that they of interest with the of this This by of to B. E. to E. and B. Z. and are by a from the Z. C. A. E. and B. B. Z. E. and B. B. Z. and B. B. Z. and Z. E. and B. B. and B. with

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