IHF Site Research Articles

A national heart failure initiative is associated with improved use of quadruple guideline-directed medical therapy

Abstract Background Heart Failure (HF) is projected to impact over 8 million people in the US by 2030. The 2022 AHA/ACC/HFSA Guideline for the Management of HF recommend four foundational medication classes for treating HFrEF patients: RAAS inhibitors, preferably angiotensin receptor/neprilysin inhibitor (ARNi), evidence-based beta-blockers (EBBB), sodium-glucose cotransporter-2 inhibitor (SGLT2i) and mineralocorticoid receptor antagonist (MRA) referred to as Quadruple Guideline-Directed Medical Therapy (Q-GDMT). Q-GDMT has shown an estimated 73% relative and 26% absolute reduction in all-cause mortality over 2 years. Yet, despite remarkable clinical potential, these therapies are patently underutilized in clinical practice. It is unknown whether an implementation science strategy may enhance adherence to Q-GDMT for HF. Methods IMPLEMENT-HF (IHF) is a national HF quality improvement initiative composed of a multidisciplinary learning collaborative and resource repository across seven U.S regions. IHF promotes adherence to Q-GDMT by focusing on early adoption and rapid adherence to prescribing at hospital discharge and within 30 days post discharge. Multi-disciplinary participation in the learning collaborative includes pharmacy, provider groups, nursing, quality, social services, outpatient clinics and others. Shared interventions to promote Q-GDMT include formulary changes (e.g., adding SGLT2i), optimizing electronic health record tools (disease-specific order sets, dot phrases), clinician education, and user-friendly patient resources to address medication access. IHF sites receive educational materials, including webinars, learning modules, pocket guideline booklets, and reminder cards to reinforce Q-GDMT. IHF participating hospitals collect Q-GDMT data at discharge and at 30 days post discharge in Get With The Guidelines®-Heart Failure (GWTG-HF). These data were analyzed from baseline (Q1 Q2 2021 aggregate) through Q3 2022. Results Data from 71 IHF participating sites of 13,858 HFrEF patient hospitalization episodes (median age 69, 34% females, median LVEF 24%), demonstrated Q-GDMT prescription at discharge increased from 14% at baseline to 47% in Q3 2022 (p value <0.001). 30-day post discharge data from 52 IHF sites demonstrated an increase in Q-GDMT from 9% at baseline to 58% in Q3 2022 (p value <0.001) as seen in Figure 1. Conclusions Among sites participating in IHF, the use of an implementation science strategy suggest a rapid increase in prescribing Q-GDMT for eligible HFrEF patients at discharge and 30-days post discharge. This multi-region quality improvement initiative demonstrates the potential for this learning collaborative model to drive improvement in adherence to Q-GDMT prescribing, using shared implementation strategies to overcome barriers. Ongoing study will assess continued compliance and clinical outcomes with Q-GDMT beyond the timeframe of this analysis.Quadruple Medication Therapy baseline-Q3

Interactions of replication initiator RctB with single- and double-stranded DNA in origin opening of Vibrio cholerae chromosome 2.

Studies of bacterial chromosomes and plasmids indicate that their replication initiator proteins bind to origins of replication at many double-stranded sites and also at AT-rich regions where single-stranded DNA is exposed during origin opening. Single-strand binding apparently promotes origin opening by stabilizing an open structure, but how the initiator participates in this process and the contributions of the several binding sites remain unclear. Here, we show that the initiator protein of Vibrio cholerae specific to chromosome 2 (Chr2) also has single-strand binding activity in the AT-rich region of its origin. Binding is strand specific, depends on repeats of the sequence 5'ATCA and is greatly stabilized in vitro by specific double-stranded sites of the origin. The stability derives from the formation of ternary complexes of the initiator with the single- and double-stranded sites. An IHF site lies between these two kinds of sites in the Chr2 origin and an IHF-induced looping out of the intervening DNA mediates their interaction. Simultaneous binding to two kinds of sites in the origin appears to be a common mechanism by which bacterial replication initiators stabilize an open origin.

IHF‐dependent activation of P1 plasmid origin by DnaA

In bacteria, many DNA-protein interactions that initiate transcription, replication and recombination require the mediation of DNA architectural proteins such as IHF and HU. For replication initiation, plasmid P1 requires three origin binding proteins: the architectural protein HU, a plasmid-specific initiator, RepA, and the Escherichia coli chromosomal initiator, DnaA. The two initiators bind in the origin of replication to multiple sites, called iterons and DnaA boxes respectively. We show here that all five known DnaA boxes can be deleted from the plasmid origin provided the origin is extended by about 120 bp. The additional DNA provides an IHF site and most likely a weak DnaA binding site, because replacing the putative site with an authentic DnaA box enhanced plasmid replication in an IHF-dependent manner. IHF most likely brings about interactions between distally bound DnaA and RepA by bending the intervening DNA. The role of IHF in activating P1 origin by allowing DnaA binding to a weak site is reminiscent of the role the protein plays in initiating the host chromosomal replication.

DNA bending in the Sin recombination synapse: functional replacement of HU by IHF

The serine recombinase Sin requires a non-specific DNA-bending protein such as Hbsu for activity at its recombination site resH. Hbsu, and Sin subunits bound at site II of resH, together regulate recombination, ensuring selectivity for directly repeated resH sites by specifying assembly of an intertwined synapse. To investigate the role of the DNA-bending protein in defining the architecture of the synapse, we constructed a chimaeric recombination site (resF) which allows Hbsu to be substituted by IHF, binding specifically between site I (the crossover site) and site II. Two Sin dimers and one IHF dimer can bind together to the closely adjoining sites in resF, forming folded complexes. The precise position of the IHF site within the site I-site II spacer determines the conformation of these complexes, and also the reactivity of the resF sites in recombination assays. The data suggest that a sharp bend with a specific geometry is required in the spacer DNA, to bring the Sin dimers at sites I and II together in the correct relative orientation for synapse assembly and regulation, consistent with our model for a highly condensed synapse in which Hbsu/IHF has a purely architectural function.

Cooperative interactions between bacteriophage P2 integrase and its accessory factors IHF and Cox

Bacteriophage P2 integrase (Int) mediates site-specific recombination leading to integration or excision of the phage genome in or out of the bacterial chromosome. Int belongs to the large family of tyrosine recombinases that have two different DNA recognition motifs binding to the arm and core sites, respectively, which are located within the phage attachment sites ( attP). In addition to the P2 integrase, the accessory proteins Escherichia coli IHF and P2 Cox are needed for recombination. IHF is a structural protein needed for integration and excision by bending the DNA. As opposed to λ, only one IHF site is found in P2 attP. P2 Cox controls the direction of recombination by inhibiting integration but being required for excision. In this work, the effects of accessory proteins on the capacity of Int to bind to its DNA recognition sequences are analyzed using electromobility shifts. P2 Int binds with low affinity to the arm site, and this binding is greatly enhanced by IHF. The arm binding domain of Int is located at the N-terminus. P2 Int binds with high affinity to the core site, and this binding is also enhanced by IHF. The fact that the cooperative binding of Int and IHF is strongly reduced by lengthening the distance between the IHF and core binding sites indicates that the distance between these sites may be important for cooperative binding. The Int and Cox proteins also bind cooperatively to attP.

Transcription activation by remodelling of a nucleoprotein assembly: the role of NarL at the FNR-dependent Escherichia coli nir promoter.

Expression from the Escherichia coli nir promoter is co-dependent on both the FNR protein (an anaerobically triggered transcription activator) and NarL or NarP proteins (transcription activators triggered by nitrite and nitrate). We found previously that FNR binds to a site centred at position - 41.5 at the nir promoter, but that FNR-dependent activation is repressed by IHF binding to a site centred at position -88 (IHF I) and Fis binding to sites centred at positions -142 (Fis I) and +23 (Fis II). Here, we have studied the binding of purified IHF, Fis and FNR to the nir promoter in vitro. Our results show that the nir promoter contains a second IHF site at position -115 (IHF II) and a third Fis site at position -97 (Fis III), and that IHF, Fis and FNR can bind together to form multiprotein complexes. Surprisingly, IHF binding at the IHF II site increases FNR-dependent activation by decreasing the repression mediated by IHF and Fis binding at the other sites. In previous work, we found that NarL or NarP activates the nir promoter by binding to a site centred at position -69.5 and counteracting the repressive effects of IHF and Fis. We now show that NarL can displace IHF bound at the IHF I site, but IHF is unable to displace bound NarL. We suggest that NarL interferes with IHF binding at the nir promoter by distorting the minor groove at its target site, and we argue that the resulting activation by NarL results from remodelling of the local nucleoprotein structure to facilitate FNR-dependent transcription.

Biochemical Investigations of Control of Replication Initiation of Plasmid R6K

The mechanistic basis of control of replication initiation of plasmid R6K was investigated by addressing the following questions. What are the biochemical attributes of mutations in the pi initiator protein that caused loss of negative control of initiation? Did the primary control involve only initiator protein-ori DNA interaction or did it also involve protein-protein interactions between pi and several host-encoded proteins? Mutations at two different regions of the pi-encoding sequence individually caused some loss of negative control as indicated by a relatively modest increase in copy number. However, combinations of the mutation P42L, which caused loss of DNA looping, with those located in the region between the residues 106 and 113 induced a robust enhancement of copy number. These mutant forms promoted higher levels of replication in vitro in a reconstituted system consisting of 22 purified proteins. The mutant forms of pi were susceptible to pronounced iteron-induced monomerization in comparison with the WT protein. As contrasted with the changes in DNA-protein interaction, we found no detectable differences in protein-protein interaction between wild type pi with DnaA, DnaB helicase, and DnaG primase on one hand and between the high copy mutant forms and the same host proteins on the other. The DnaG-pi interaction reported here is novel. Taken together, the results suggest that both loss of negative control due to iteron-induced monomerization of the initiator and enhanced iteron-initiator interaction appear to be the principal causes of enhanced copy number.

The positive and negative regulation of Tn10 transposition by IHF is mediated by structurally asymmetric transposon arms.

The Tn10 transpososome has symmetrical components on either side: there are two transposon ends each of which has binding sites for a monomer of transposase and an IHF heterodimer. The DNA bending activity of IHF stimulates assembly of an intermediate with tightly folded transposon ends in which transposase has additional 'subterminal' DNA contacts, located distal to the IHF site. These subterminal contacts are required to activate later steps in the reaction. Quantitative hydroxyl radical footprinting and gel retardation unfolding experiments show that the transpososome is fundamentally asymmetric, despite having identical components on either side. Major differences between the transposon ends define alpha and beta sides of the complex. IHF can dissociate from the transposon arm on the beta side of the complex in the absence of metal ion. However, IHF is locked onto the alpha side of the complex, probably by the subterminal transposase contacts, until released by a metal ion-dependent conformational change. Later in the reaction, IHF inhibits target interactions. Using a very short transposon arm, target interactions are demonstrated at a saturating IHF concentration. This suggests that inhibition of target interactions is due to steric hindrance of the target binding site by a single IHF-folded transposon arm.

Protein-DNA contacts and conformational changes in the Tn10 transpososome during assembly and activation for cleavage.

IHF or supercoiling is required early in Tn10 transposition, but at later stages they inhibit the reaction in a classic homeostatic loop. We investigated the mechanism of transpososome assembly and regulation using hydroxyl radical DNA protection and interference. We present a three-dimensional molecular model for the IHF-bent end of Tn10 wrapped around a transposase core. Contacts span some 80 bp at the transposon end, but after assembly of an active complex containing metal ion, most contacts become dispensable. These include transposase contacts beyond the IHF site that chaperone assembly of the complex and are needed for efficient cleavage. Single and double-end breaks do not affect the complex but divalent metal ions promote large conformational changes at bp +1 and the flanking DNA.

A quantitative UV laser footprinting analysis of the interaction of IHF with specific binding sites: re-evaluation of the effective concentration of IHF in the cell

The integration host factor (IHF) of Escherichia coli is a major nucleoid-associated protein that binds to specific sites on DNA. Using gel retardation and competition experiments we have estimated that in vitro IHF binds specific sites 1000–10,000 times more tightly than non-specific, chromosomal DNA. We have analyzed the in vitro and in vivo interaction of IHF with three specific binding sites using UV laser footprinting. Because there is a strict correspondence between the intensity of the footprinting signal and the occupancy of a site, we can correlate in vitro association constants with in vivo site occupancy. From the fractional occupancy of various ihf sites in vivo, we then estimate the amount of free IHF in the cell. Exponentially growing cells contain only about 0.7 nM of free IHF, a value 20-fold smaller than the one previously deduced from DMS footprinting. As a consequence low affinity sites are only partially occupied and strong binding sites reach semi-saturation. In stationary phase the concentration of free IHF in the cell increases about sevenfold. These results show that only a very small fraction of total IHF is free in solution. Given the affinity of IHF for non-specific DNA our data imply that a large part of chromosomal DNA is accessible to IHF, and that IHF is a major contributor to chromosomal DNA condensation. The in vivo UV-laser footprinting method is of general interest, because it allows the measurement and the comparison of DNA-protein interactions in vitro and in vivo.

In vitro interaction of the IHF-like proteins Acinetobacter junii and Proteus vulgaris with ihf sites.

The ability of the IHF-like proteins of Acinetobacter junii and Proteus vulgaris to interact with the H' attP and pR' ihf sites of lambda DNA was investigated. IHF from A. junii and P. vulgaris was found to bind the examined ihf sites in a way similar to IHF from Escherichia coli as shown by gel mobility shift DNA binding assays and footprinting analysis. The three IHF proteins bound to the pR' ihf site that overlaps the-35 region of that promoter and in vitro repression of transcription by each IHF was observed. These results confirm that IHF-like proteins from Gram-negative bacteria can recognize the same specific DNA sequences and appear to be important in regulation of transcription.

In vitro interaction of the IHF-like proteins Acinetobacter junii and Proteus vulgaris with ihf sites

The ability of the IHF-like proteins of Acinetobacter junii and Proteus vulgaris to interact with the H′ attP and p R′ ihf sites of λ DNA was investigated. IHF from A. junii and P. vulgaris was found to bind the examined ihf sites in a way similar to IHF from Escherichia coli as shown by gel mobility shift DNA binding assays and footprinting analysis. The three IHF proteins bound to the p R′ ihf site that overlaps the −35 region of that promoter and in vitro repression of transcription by each IHF was observed. These results confirm that IHF-like proteins from Gram-negative bacteria can recognize the same specific DNA sequences and appear to be important in regulation of transcription.

Deletion analysis of the fis promoter region in Escherichia coli: antagonistic effects of integration host factor and Fis.

Fis is a small DNA-binding and -bending protein in Escherichia coli that is involved in several different biological processes, including stimulation of specialized DNA recombination events and regulation of gene expression. fis protein and mRNA levels rapidly increase during early logarithmic growth phase in response to a nutritional upshift but become virtually undetectable during late logarithmic and stationary phases. We present evidence that the growth phase-dependent fis expression pattern is not determined by changes in mRNA stability, arguing in favor of regulation at the level of transcription. DNA deletion analysis of the fis promoter (fis P) region indicated that DNA sequences from -166 to -81, -36 to -26, and +107 to +366 relative to the transcription start site are required for maximum expression. A DNA sequence resembling the integration host factor (IHF) binding site centered approximately at -114 showed DNase I cleavage protection by IHF. In ihf cells, maximum cellular levels of fis mRNA were decreased more than 3-fold and transcription from fis P on a plasmid was decreased about 3.8-fold compared to those in cells expressing wild-type IHF. In addition, a mutation in the ihf binding site resulted in a 76 and 61% reduction in transcription from fis P on a plasmid in the presence or absence of Fis, respectively. Insertions of 5 or 10 bp between this ihf site and fis P suggest that IHF functions in a position-dependent manner. We conclude that IHF plays a role in stimulating transcription from fis P by interacting with a site centered approximately at -114 relative to the start of transcription. We also showed that although the fis P region contains six Fis binding sites, Fis site II (centered at -42) played a predominant role in autoregulation, Fis sites I and III (centered at +26 and -83, respectively) seemingly played smaller roles, and no role in negative autoregulation could be attributed to Fis sites IV, V, and VI (located upstream of site III). The fis P region from -36 to +7, which is not directly regulated by either IHF or Fis, retained the characteristic fis regulation pattern in response to a nutritional upshift.

The integration host factor-DNA complex upstream of the early promoter of bacteriophage Mu is functionally symmetric.

Inversion of the ihf site in the promoter region of the early promoter of bacteriophage Mu did not influence the integration host factor (IHF)-mediated functions. IHF bound to this inverted site could counteract H-NS-mediated repression, directly activate transcription, and support lytic growth of bacteriophage Mu. This implies that the IHF heterodimer and its asymmetrical binding site form a functionally symmetrical complex.

Integration host factor alleviates the H-NS-mediated repression of the early promoter of bacteriophage Mu.

Integration host factor (IHF), which is a histone-like protein, has been shown to positively regulate transcription in two different ways. It can either help the formation of a complex between a transcription factor and RNA polymerase or it can itself activate RNA polymerase without the involvement of other transcription factors. In this study, we present a third mechanism for IHF-stimulated gene expression, by counteracting the repression by another histone-like protein, H-NS. The early (Pe) promoter of bacteriophage Mu is specifically inhibited by H-NS, both in vivo and in vitro. For this inhibition, H-NS binds to a large DNA region overlapping the Pe promoter. Binding of IHF to a binding site just upstream of Pe alleviates the H-NS-mediated repression of transcription. This same ihf site is also involved in the direct activation of Pe by IHF. In contrast to the direct activation by IHF, however, the alleviating effect of IHF appears not to be dependent on the relevant position of the ihf site on the DNA helix, and it also does not require the presence of the C-terminal domain of the alpha subunit of RNA polymerase. Footprint analysis shows that binding of IHF to the ihf site destabilizes the interaction of H-NS with the DNA, not only in the IHF-binding region but also in the DNA regions flanking the ihf site. These results suggest that IHF disrupts a higher-order nucleoprotein complex that is formed by H-NS and the DNA.

Purification of IHF-like protein from gram-negative bacteria in one chromatographic step.

We describe a fast and very efficient method of purification which yields highly purified integration host factor-like proteins in one chromatographic step. IHF-like proteins from Acinetobacter junii or Proteus vulgaris are each an alpha beta heterodimer (subunits of 10 and 11 kDa) similar to the IHF of Escherichia coli when analyzed by polyacrylamide gel electrophoresis. The purified IHF are able to bind to the same ihf sites as IHF of E. coli. The results presented confirm that IHF is conserved during evolution in gram-negative bacteria.

Mu transposase-stimulated illegitimate recombination of Tn 3kan- and IS 101-containing plasmids

The transposable bacteriophage Mu and the mobile genetic elements Tn 3 and IS 101 replicatively transpose to random target sites, produce 5 bp target site duplications, and contain the sequence 5′-PuCGAAPu-3′ starting at bo 21 from their ends. The presence of these shared characteristics, plus the fact that Mu transposase can specifically bind to the termini of Tn 3 and IS 101 in vitro, suggests that the elements may be evolutionarily conserved and retain some functional capacity to transpose each other's DNA. To examine this proposition, in vivo transposition-mating assays were performed and demonstrated that mu transposase stimulated the formation of recA-independent recombination products between Tn 3kan- or IS 101-containing plasmids and a target plasmid (pOX38cam) up to 200-fold. However, when transferred to recA + hosts, these recA-independent products yielded resolution products suggestive of illegitimate recombination, as similar recombination and resolution products were generated, at reduced frequencies, in the absence of Mu transposase. Thus, Mu transposase may stimulate a host-mediated, recA-independent illegitimate recombination reaction. As adjacent pSC101 sequence, including a formerly unknown but functional IHF site (bp 2238–2251), were required for Mu transposase-stimulated IS 101 illegitimate recombination, IHF may be one of the putative host factors involved in these recombination reactions.

Participation of the Flank Regions of the Integration Host Factor Protein in the Specificity and Stability of DNA Binding

The heterodimeric integration host factor (IHF) protein is a site-specific DNA-binding protein from Escherichia coli that strongly bends the DNA. It has been proposed (Yang, C., and Nash, H.A. (1989) Cell 57, 869-880; Granston, A. E., and Nash, H. A. (1993) J. Mol. Biol 234, 45-59; Lee, E. C., Hales, L. M., Gumport, R. I., and Gardner, J. F. (1992) EMBO J. 11, 305-313) that the wrapping of the DNA around the protein is stabilized through interactions between the flanks of the protein and the DNA. In order to elucidate which domains of the IHF protein are involved in these interactions, we have constructed mutant proteins in which the C-terminal part of one of the subunits has been deleted. We observed that the C-terminal alpha 3 helix of HimD is involved in the stability of DNA binding, but not in the specificity. In contrast the corresponding alpha 3 helix of HimA is essential for the sequence specificity, since an IHF mutant lacking this domain only binds to the DNA in a non-specific way. The possible role of the two C-terminal alpha-helical structures in complex formation will be discussed. We also examined the properties of an IHF mutant that has an amino acid substitution between beta sheets beta 1 and beta 2 of the HimD subunit (R46H). The occupancy of the ihf site by the mutant and wild type proteins differ in the 3' part of the ihf site and as a result the bend introduced in the DNA by the mutant protein is less pronounced. We propose that the arginine 46 in the HimD subunit is in vicinity of the TTR region of the consensus and that through contacts within the minor groove the DNA bend introduced by IHF is stabilized.

Binding of Escherichia coli integration host factor (IHF) to the origin segment of p15A plasmid.

The integration host factor (IHF) is a sequence-specific, histone-like, multi-functional DNA-binding and -bending protein of Escherichia coli. Characterization and functional analysis of this protein has been carried out mainly in bacteriophage lambda and other mobile genetic elements. In this paper we report data concerning the binding of IHF protein to the plasmid orip15A region. IHF binds to the single site of the DNA fragment containing the orip15A, as shown by the gel mobility shift assays and footprinting experiment. On the basis of the ihf consensus sequences published, we have been able to identify one sequence of putative ihf site into the orip15A sequence with two mismatches in relation to the consensus sequence of Kur et al., 1989, Gene 81, 1-15. One ihf binding site was also found in the oriColE1 region sequence with three mismatches in relation to this consensus sequence.

Specific interaction of IHF with RIBs, a class of bacterial repetitive DNA elements located at the 3' end of transcription units.

The prokaryotic REP (repetitive extragenic palindromes) or PU (palindromic units) sequences are often associated with other repetitive elements, forming arrangements which have been called 'BIMEs' (bacterial interspersed mosaic elements). It is estimated that the Escherichia coli chromosome carries approximately 300-500 BIMEs, whose biological role is at present unknown. We have identified a family of BIMEs consisting of two converging REP sequences flanking a 35 bp conserved segment which carries a static DNA bend and a binding site for IHF, the integration host factor of E.coli. We estimate that the E.coli genome harbors approximately 100 copies of this module, which we name 'RIB' (reiterative ihf BIME). We have analyzed by gel retardation and by footprinting the in vitro interaction of IHF with individual RIBs, and shown that the protein binds strongly and specifically to their center. We have also demonstrated binding of IHF to the chromosomal population of RIBs, using a new approach which combines two-dimensional bandshift and Southern blotting. RIB elements are at the end of transcription units, and thus define a new class of ihf sites. Possible implications for genome structure and DNA topology are discussed.